Modulation of factor 11 expression

ABSTRACT

Disclosed herein are antisense compounds and methods for decreasing Factor 11 and treating or preventing thromboembolic complications in an individual in need thereof. Examples of disease conditions that can be ameliorated with the administration of antisense compounds targeted to Factor 11 include thrombosis, embolism, and thromboembolism, such as, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Antisense compounds targeting Factor 11 can also be used as a prophylactic treatment to prevent individuals at risk for thrombosis and embolism.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0107SEQ.xml created Jan. 3, 2023, which is 271 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Embodiments of the present invention provide methods, compounds, andcompositions for reducing expression of Factor 11 mRNA and protein in ananimal. Such methods, compounds, and compositions are useful to treat,prevent, or ameliorate thromboembolic complications.

BACKGROUND OF THE INVENTION

The circulatory system requires mechanisms that prevent blood loss, aswell as those that counteract inappropriate intravascular obstructions.Generally, coagulation comprises a cascade of reactions culminating inthe conversion of soluble fibrinogen to an insoluble fibrin gel. Thesteps of the cascade involve the conversion of an inactive zymogen to anactivated enzyme. The active enzyme then catalyzes the next step in thecascade.

Coagulation Cascade

The coagulation cascade may be initiated through two branches, thetissue factor pathway (also “extrinsic pathway”), which is the primarypathway, and the contact activation pathway (also “intrinsic pathway”).

The tissue factor pathway is initiated by the cell surface receptortissue factor (TF, also referred to as factor III), which is expressedconstitutively by extravascular cells (pericytes, cardiomyocytes, smoothmuscle cells, and keratinocytes) and expressed by vascular monocytes andendothelial cells upon induction by inflammatory cytokines or endotoxin.(Drake et al., Am J Pathol 1989, 134:1087-1097). TF is the high affinitycellular receptor for coagulation factor VIIa, a serine protease. In theabsence of TF, VIIa has very low catalytic activity, and binding to TFis necessary to render VIIa functional through an allosteric mechanism.(Drake et al., Am J Pathol 1989, 134:1087-1097). The TF-VIIa complexactivates factor X to Xa. Xa in turn associates with its co-factorfactor Va into a prothrombinase complex which in turn activatesprothrombin, (also known as factor II or factor 2) to thrombin (alsoknown as factor IIa, or factor 2a). Thrombin activates platelets,converts fibrinogen to fibrin and promotes fibrin cross-linking byactivating factor XIII, thus forming a stable plug at sites where TF isexposed on extravascular cells. In addition, thrombin reinforces thecoagulation cascade response by activating factors V and VIII.

The contact activation pathway is triggered by activation of factor XIIto XIIa. Factor XIIa converts XI to XIa, and XIa converts IX to IXa. IXaassociates with its cofactor VIIIa to convert X to Xa. The two pathwaysconverge at this point as factor Xa associates factor Va to activateprothrombin (factor II) to thrombin (factor IIa).

Inhibition of Coagulation.

At least three mechanisms keep the coagulation cascade in check, namelythe action of activated protein C, antithrombin, and tissue factorpathway inhibitor. Activated protein C is a serine protease thatdegrades cofactors Va and VIIIa. Protein C is activated by thrombin withthrombomodulin, and requires coenzyme Protein S to function.Antithrombin is a serine protease inhibitor (serpin) that inhibitsserine proteases: thrombin, Xa, XIIa, XIa and IXa. Tissue factor pathwayinhibitor inhibits the action of Xa and the TF-VIIa complex. (Schwartz AL et al., Trends Cardiovasc Med. 1997; 7:234-239.)

Disease

Thrombosis is the pathological development of blood clots, and anembolism occurs when a blood clot migrates to another part of the bodyand interferes with organ function. Thromboembolism may cause conditionssuch as deep vein thrombosis, pulmonary embolism, myocardial infarction,and stroke. Significantly, thromboembolism is a major cause of morbidityaffecting over 2 million Americans every year. (Adcock et al. AmericanJournal of Clinical Pathology. 1997; 108:434-49). While most cases ofthrombosis are due to acquired extrinsic problems, for example, surgery,cancer, immobility, some cases are due to a genetic predisposition, forexample, antiphospholipid syndrome and the autosomal dominant condition,Factor V Leiden. (Bertina R M et al. Nature 1994; 369:64-67.)

Treatment.

The most commonly used anticoagulants, warfarin, heparin, and lowmolecular weight heparin (LMWH) all possess significant drawbacks.

Warfarin is typically used to treat patients suffering from atrialfibrillation. The drug interacts with vitamin K-dependent coagulationfactors which include factors II, VII, IX and X. Anticoagulant proteinsC and S are also inhibited by warfarin. Drug therapy using warfarin isfurther complicated by the fact that warfarin interacts with othermedications, including drugs used to treat atrial fibrillation, such asamiodarone. Because therapy with warfarin is difficult to predict,patients must be carefully monitored in order to detect any signs ofanomalous bleeding.

Heparin functions by activating antithrombin which inhibits boththrombin and factor X. (Bjork I, Lindahl U. Mol Cell Biochem. 1982 48:161-182.) Treatment with heparin may cause an immunological reactionthat makes platelets aggregate within blood vessels that can lead tothrombosis. This side effect is known as heparin-inducedthrombocytopenia (HIT) and requires patient monitoring. Prolongedtreatment with heparin may also lead to osteoporosis. LMWH can alsoinhibit Factor 2, but to a lesser degree than unfractioned heparin(UFH). LMWH has been implicated in the development of HIT.

Thus, current anticoagulant agents lack predictability and specificityand, therefore, require careful patient monitoring to prevent adverseside effects, such as bleeding complications. There are currently noanticoagulants which target only the intrinsic or extrinsic pathway.

SUMMARY OF THE INVENTION

Provided herein are methods, compounds, and compositions for modulatingexpression of Factor 11 mRNA and protein. In certain embodiments, Factor11 specific inhibitors modulate expression of Factor 11 mRNA andprotein. In certain embodiments, Factor 11 specific inhibitors arenucleic acids, proteins, or small molecules.

In certain embodiments, modulation can occur in a cell or tissue. Incertain embodiments, the cell or tissue is in an animal. In certainembodiments, the animal is a human. In certain embodiments, Factor 11mRNA levels are reduced. In certain embodiments, Factor 11 proteinlevels are reduced. Such reduction can occur in a time-dependent manneror in a dose-dependent manner.

Also provided are methods, compounds, and compositions useful forpreventing, treating, and ameliorating diseases, disorders, andconditions. In certain embodiments, such diseases, disorders, andconditions are thromboembolic complications. Such thromboemboliccomplications include the categories of thrombosis, embolism, andthromboembolism. In certain embodiments such thromboemboliccomplications include deep vein thrombosis, pulmonary embolism,myocardial infarction, and stroke.

Such diseases, disorders, and conditions can have one or more riskfactors, causes, or outcomes in common. Certain risk factors and causesfor development of a thromboembolic complication include immobility,surgery (particularly orthopedic surgery), malignancy, pregnancy, olderage, use of oral contraceptives, atrial fibrillation, previousthromboembolic complication, chronic inflammatory disease, and inheritedor acquired prothrombotic clotting disorders. Certain outcomesassociated with development of a thromboembolic complication includedecreased blood flow through an affected vessel, death of tissue, anddeath.

In certain embodiments, methods of treatment include administering aFactor 11 specific inhibitor to an individual in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout in the disclosure herein are incorporated byreference for the portions of the document discussed herein, as well asin their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the 2′ position of a furosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to Factor 11 is an activepharmaceutical agent.

“Active target region” or “target region” means a region to which one ormore active antisense compounds is targeted. “Active antisensecompounds” means antisense compounds that reduce target nucleic acidlevels or protein levels.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an individual,and includes, but is not limited to administering by a medicalprofessional and self-administering.

“Amelioration” refers to a lessening of at least one indicator, sign, orsymptom of an associated disease, disorder, or condition. The severityof indicators may be determined by subjective or objective measures,which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antidote compound” refers to a compound capable of decreasing theintensity or duration of any antisense-mediated activity.

“Antidote oligonucleotide” means an antidote compound comprising anoligonucleotide that is complementary to and capable of hybridizing withan antisense compound.

“Antidote protein” means an antidote compound comprising a peptide.

“Antibody” refers to a molecule characterized by reacting specificallywith an antigen in some way, where the antibody and the antigen are eachdefined in terms of the other. Antibody may refer to a complete antibodymolecule or any fragment or region thereof, such as the heavy chain, thelight chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

“Antisense inhibition” means reduction of target nucleic acid levels ortarget protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

“Coagulation factor” means any of factors I, II, III, IV, V, VII, VIII,IX, X, XI, XII, XIII, or TAFI in the blood coagulation cascade.“Coagulation factor nucleic acid” means any nucleic acid encoding acoagulation factor. For example, in certain embodiments, a coagulationfactor nucleic acid includes, without limitation, a DNA sequenceencoding a coagulation factor (including genomic DNA comprising intronsand exons), an RNA sequence transcribed from DNA encoding a coagulationfactor, and an mRNA sequence encoding a coagulation factor. “Coagulationfactor mRNA” means an mRNA encoding a coagulation factor protein.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” means the amount of active pharmaceutical agentsufficient to effectuate a desired physiological outcome in anindividual in need of the agent. The effective amount may vary amongindividuals depending on the health and physical condition of theindividual to be treated, the taxonomic group of the individuals to betreated, the formulation of the composition, assessment of theindividual's medical condition, and other relevant factors.

“Factor 11 nucleic acid” or “Factor XI nucleic acid” or “F 11 nucleicacid” or “F XI nucleic acid” means any nucleic acid encoding Factor 11.For example, in certain embodiments, a Factor 11 nucleic acid includes aDNA sequence encoding Factor 11, an RNA sequence transcribed from DNAencoding Factor 11 (including genomic DNA comprising introns and exons),and an mRNA sequence encoding Factor 11. “Factor 11 mRNA” means an mRNAencoding a Factor 11 protein.

“Factor 11 specific inhibitor” refers to any agent capable ofspecifically inhibiting the expression of Factor 11 mRNA and/or Factor11 protein at the molecular level. For example, Factor 11 specificinhibitors include nucleic acids (including antisense compounds),peptides, antibodies, small molecules, and other agents capable ofinhibiting the expression of Factor 11 mRNA and/or Factor 11 protein. Incertain embodiments, by specifically modulating Factor 11 mRNAexpression and/or Factor 11 protein expression, Factor 11 specificinhibitors may affect other components of the coagulation cascadeincluding downstream components. Similarly, in certain embodiments,Factor 11 specific inhibitors may affect other molecular processes in ananimal.

“Factor 11 specific inhibitor antidote” means a compound capable ofdecreasing the effect of a Factor 11 specific inhibitor. In certainembodiments, a Factor 11 specific inhibitor antidote is selected from aFactor 11 peptide; a Factor 11 antidote oligonucleotide, including aFactor 11 antidote compound complementary to a Factor 11 antisensecompound; and any compound or protein that affects the intrinsic orextrinsic coagulation pathway.

“Fully complementary” or “100% complementary” means each nucleobase of afirst nucleic acid has a complementary nucleobase in a second nucleicacid. In certain embodiments, a first nucleic acid is an antisensecompound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap segment” andthe external regions may be referred to as “wing segments.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Identifying an animal at risk for thromboembolic complications” meansidentifying an animal having been diagnosed with a thromboemboliccomplication or identifying an animal predisposed to develop athromboembolic complication. Individuals predisposed to develop athromboembolic complication include those having one or more riskfactors for thromboembolic complications including immobility, surgery(particularly orthopedic surgery), malignancy, pregnancy, older age, useof oral contraceptives, and inherited or acquired prothrombotic clottingdisorders. Such identification may be accomplished by any methodincluding evaluating an individual's medical history and standardclinical tests or assessments.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e. aphosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase”means the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U).

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, or modifiednucleobase. A “modified nucleoside” means a nucleoside having,independently, a modified sugar moiety or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising amodified internucleoside linkage, a modified sugar, or a modifiednucleobase.

“Modified sugar” refers to a substitution or change from a naturalsugar.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids, double-stranded nucleic acids,small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units.Nucleotide mimetic includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage). Sugar surrogate overlaps with the slightly broader termnucleoside mimetic but is intended to indicate replacement of the sugarunit (furanose ring) only. The tetrahydropyranyl rings provided hereinare illustrative of an example of a sugar surrogate wherein the furanosesugar group has been replaced with a tetrahydropyranyl ring system.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition may comprise one or more active pharmaceutical agents and asterile aqueous solution.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage (P=S) is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prevent” refers to delaying or forestalling the onset or development ofa disease, disorder, or condition for a period of time from minutes toindefinitely. Prevent also means reducing risk of developing a disease,disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Side effects” means physiological responses attributable to a treatmentother than the desired effects. In certain embodiments, side effectsinclude injection site reactions, liver function test abnormalities,renal function abnormalities, liver toxicity, renal toxicity, centralnervous system abnormalities, myopathies, and malaise. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an antisenseoligonucleotide and a target nucleic acid to induce a desired effect,while exhibiting minimal or no effects on non-target nucleic acids underconditions in which specific binding is desired, i.e. underphysiological conditions in the case of in vivo assays and therapeutictreatments.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Thromboembolic complication” means any disease, disorder, or conditioninvolving an embolism caused by a thrombus. Examples of such diseases,disorders, and conditions include the categories of thrombosis,embolism, and thromboembolism. In certain embodiments, such diseasedisorders, and conditions include deep vein thrombosis, pulmonaryembolism, myocardial infarction, and stroke.

“Treat” refers to administering a pharmaceutical composition to effectan alteration or improvement of a disease, disorder, or condition.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Certain Embodiments

Embodiments of the present invention provide methods, compounds, andcompositions for decreasing Factor 11 mRNA and protein expression.

Embodiments of the present invention provide methods, compounds, andcompositions for the treatment, prevention, or amelioration of diseases,disorders, and conditions associated with Factor 11 in an individual inneed thereof. Also contemplated are methods and compounds for thepreparation of a medicament for the treatment, prevention, oramelioration of a disease, disorder, or condition associated with Factor11. Factor 11 associated diseases, disorders, and conditions includethromboembolic complications such as thrombosis, embolism,thromboembolism, deep vein thrombosis, pulmonary embolism, myocardialinfarction, and stroke.

Embodiments of the present invention provide a Factor 11 specificinhibitor for use in treating, preventing, or ameliorating a Factor 11associated disease. In certain embodiments, Factor 11 specificinhibitors are nucleic acids (including antisense compounds), peptides,antibodies, small molecules, and other agents capable of inhibiting theexpression of Factor 11 mRNA and/or Factor 11 protein.

In certain embodiments of the present invention, Factor 11 specificinhibitors are peptides or proteins, such as, but not limited to, alpha1 protease inhibitors, antithrombin III, C1 inhibitors, and alpha 2plasmin inhibitors as described in J Clin Invest 1982, 69:844-852; alpha1 antitrypsin (alpha 1AT) as described in Thromb Res 1987, 48:145-151;Factor 11 peptide inhibitors as described in USPPN 2008/021998 and Blood1998, 92:4198-206; MAP4-RGKWC as described in Thromb Res 2001,104:451-465; beta 2 GPI as described in Proc Natl Acad Sci 2004,101:3939-44; Lentinus proteinase inhibitor as described in Eur J Biochem1999, 262:915-923; protease nexin-2/amyloid beta protein precursorKunitz domain inhibitor (APPI) and antithrombin (AT) as described in JBiol Chem 2004, 279:29485-29492; and aprotinin as described in J BiolChem 2005, 280:23523-30.

In certain embodiments of the present invention, Factor 11 specificinhibitors are antibodies, such as, but not limited to, Winston-Salem(IgG3 kappa) and Baltimore (IgG1 kappa) as described in Blood 1988,72:1748-54; 5F4, 3C1, and 1F1 as described in J Biol Chem 1985,260:10714-719; monoclonal antibodies as described in Throm Haemost 1990,63:417-23; XI-5108 as described in J Thromb Haem 2006, 4:1496-1501;monoclonal antibodies 4-1 as described in Thromb Res 1986, 42:225-34;and abcixmab antibody as described in Example 19 of U.S. Pat. No.6,566,140.

In certain embodiments of the present invention, Factor 11 specificinhibitors are small molecules, such as, but not limited to, diisopropylfluorophosphates (DFP); the small molecule inhibitors as described inExamples 1-7 of USPPN 2004/0180855; and p-aminobenzamidine (pAB) asdescribed in J Biol Chem 2005, 280:23523-30.

Embodiments of the present invention provide a Factor 11 specificinhibitor, as described herein, for use in treating, preventing, orameliorating thromboembolic complications such as thrombosis, embolism,thromboembolism, deep vein thrombosis, pulmonary embolism, myocardialinfarction, and stroke.

Embodiments of the present invention provide the use of Factor 11specific inhibitors as described herein in the manufacture of amedicament for treating, ameliorating, or preventing a thromboemboliccomplication such as thrombosis, embolism, thromboembolism, deep veinthrombosis, pulmonary embolism, myocardial infarction, and stroke.

Embodiments of the present invention provide a Factor 11 specificinhibitor as described herein for use in treating, preventing, orameliorating a thromboembolic complication as described herein bycombination therapy with an additional agent or therapy as describedherein. Agents or therapies can be co-administered or administeredconcomitantly.

Embodiments of the present invention provide the use of a Factor 11specific inhibitor as described herein in the manufacture of amedicament for treating, preventing, or ameliorating a thromboemboliccomplication as described herein by combination therapy with anadditional agent or therapy as described herein. Agents or therapies canbe co-administered or administered concomitantly.

Embodiments of the present invention provide the use of a Factor 11specific inhibitor as described herein in the manufacture of amedicament for treating, preventing, or ameliorating a thromboemboliccomplication as described herein in a patient who is subsequentlyadministered an additional agent or therapy as described herein.

Embodiments of the present invention provide a kit for treating,preventing, or ameliorating a thromboembolic complication as describedherein wherein the kit comprises:

-   -   (i) a Factor 11 specific inhibitor as described herein; and        alternatively    -   (ii) an additional agent or therapy as described herein.

A kit of the present invention may further include instructions forusing the kit to treat, prevent, or ameliorate a thromboemboliccomplication as described herein by combination therapy as describedherein.

Embodiments of the present invention provide antisense compoundstargeted to a Factor 11 nucleic acid. In certain embodiments, the Factor11 nucleic acid is any of the sequences set forth in GENBANK AccessionNo. NM_000128.3 (incorporated herein as SEQ ID NO: 1), GENBANK AccessionNo. NT_022792.17, truncated from 19598000 to 19624000, (incorporatedherein as SEQ ID NO: 2), GENBANK Accession No. NM_028066.1 (incorporatedherein as SEQ ID NO: 6), exons 1-15 GENBANK Accession No. NW_001118167.1(incorporated herein as SEQ ID NO: 274).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide. In certain embodiments, the compound of theinvention comprises a modified oligonucleotide consisting of 12 to 30linked nucleosides.

In certain embodiments, the compound of the invention may comprise amodified oligonucleotide comprising a nucleobase sequence at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% complementary to an equal length portion ofSEQ ID NO: 1. In certain embodiments, the compound of the invention maycomprise a modified oligonucleotide comprising a nucleobase sequence100% complementary to an equal length portion of SEQ ID NO: 1.

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 656 to 676 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 656 to 676 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 665 to 687 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 665 to 687 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 50% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 675 to 704 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 675 to 704 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 50% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 677 to 704 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 677 to 704 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 60% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 678 to 697 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 678 to 697 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 70% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 680 to 703 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 680 to 703 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example 3and Example 30).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 683 to 702 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 683 to 702 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 90% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 738 to 759 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 738 to 759 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example 3and Example 30).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 738 to 760 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 738 to 760 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 60% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 738 to 762 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 738 to 762 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 45% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1018 to 1042 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1018 to 1042 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1062 to 1089 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1062 to 1089 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 70% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1062 to 1090 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1062 to 1090 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 60% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1062 to 1091 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 20% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1275 to 1301 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1062 to 1091 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1276 to 1301 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1062 to 1091 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example30).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1284 to 1308 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1062 to 1091 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1291 to 1317 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1062 to 1091 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 80% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

In certain embodiments, the invention provides a compound comprising amodified oligonucleotide comprising a nucleobase sequence complementaryto at least a portion of nucleobases 1275 to 1318 of SEQ ID NO: 1. Saidmodified oligonucleotide may comprise at least 8, at least 10, at least12, at least 14, at least 16, at least 18 or 20 contiguous nucleobasescomplementary to an equal length portion of nucleobases 1275 to 1318 ofSEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobasesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% complementary toan equal length portion of SEQ ID NO: 1. Said modified oligonucleotidemay comprise a nucleobase sequence 100% complementary to an equal lengthportion of SEQ ID NO: 1. Said modified oligonucleotide may achieve atleast 70% inhibition of human mRNA levels as determined using an RT-PCRassay method, optionally in HepG2 cells (e.g. as described in Example3).

Embodiments of the present invention provide compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 18, or 20 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 15 to 241.

Embodiments of the present invention provide compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 18, or 20 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 15 to 269.

Embodiments of the present invention provide compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 18, or 20 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 242 to 269.

In certain embodiments, the modified oligonucleotide comprises at least8, at least 10, at least 12, at least 14, at least 16, or at least 18nucleobases of a nucleobase sequence selected from SEQ ID NOs: 22, 31,32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64, 66, 71, 73,75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127, 129, 171, 172,174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to 232. In certainembodiments, the modified oligonucleotide comprises a nucleobasesequence selected from SEQ ID NOs: 22, 31, 32, 34, 36 to 38, 40, 41, 43,51 to 53, 55, 56, 59, 60, 64, 66, 71, 73, 75, 96, 98 to 103, 105 to 109,113 to 117, 119, 124, 127, 129, 171, 172, 174, 176, 178, 179, 181 to197, 199 to 211, and 213 to 232. In certain embodiments, the modifiedoligonucleotide consists of a nucleobase sequence selected from SEQ IDNOs: 22, 31, 32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64,66, 71, 73, 75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127,129, 171, 172, 174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to232. Said modified oligonucleotide may achieve at least 70% inhibitionof human mRNA levels as determined using an RT-PCR assay method,optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the modified oligonucleotide comprises at least8, at least 10, at least 12, at least 14, at least 16, or at least 18nucleobases of a nucleobase sequence selected from SEQ ID NOs: 22, 31,34, 37, 40, 43, 51 to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119,171, 174, 176, 179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213to 232. In certain embodiments, the modified oligonucleotide comprises anucleobase sequence selected from SEQ ID NOs: 22, 31, 34, 37, 40, 43, 51to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176,179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. Incertain embodiments, the modified oligonucleotide consists of anucleobase sequence selected from SEQ ID NOs: 22, 31, 34, 37, 40, 43, 51to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176,179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. Saidmodified oligonucleotide may achieve at least 80% inhibition of humanmRNA levels as determined using an RT-PCR assay method, optionally inHepG2 cells (e.g. as described in Example 3).

In certain embodiments, the modified oligonucleotide comprises at least8, at least 10, at least 12, at least 14, at least 16, or at least 18nucleobases of a nucleobase sequence selected from SEQ ID NOs: 31, 37,100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214 to 219, 221 to224, 226, 227, 229, and 231. In certain embodiments, the modifiedoligonucleotide comprises a nucleobase sequence selected from SEQ IDNOs: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214to 219, 221 to 224, 226, 227, 229, and 231. In certain embodiments, themodified oligonucleotide consists of a nucleobase sequence selected fromSEQ ID NOs: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209,210, 214 to 219, 221 to 224, 226, 227, 229, and 231.

Said modified oligonucleotide may achieve at least 90% inhibition ofhuman mRNA levels as determined using an RT-PCR assay method, optionallyin HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the modified oligonucleotide comprises at least8, at least 10, at least 12, at least 14, at least 16, or at least 18nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 52,53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266. In certainembodiments, the modified oligonucleotide comprises a nucleobasesequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to232, 242 to 260, and 262 to 266. In certain embodiments, the modifiedoligonucleotide consists of a nucleobase sequence selected from SEQ IDNOs: 34, 52, 53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266.Said modified oligonucleotides may achieve at least 70% inhibition ofhuman mRNA levels as determined using an RT-PCR assay method, optionallyin HepG2 cells (e.g. as described in Example 30).

In certain embodiments, the modified oligonucleotide comprises at least8, at least 10, at least 12, at least 14, at least 16, or at least 18nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 52,53, 114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to259, 264, and 265. In certain embodiments, the modified oligonucleotidecomprises a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53,114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259,264, and 265. In certain embodiments, the modified oligonucleotideconsists of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53,114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259,264, and 265. Said modified oligonucleotides may achieve at least 80%inhibition of human mRNA levels as determined using an RT-PCR assaymethod, optionally in HepG2 cells (e.g. as described in Example 30).

In certain embodiments, the modified oligonucleotide comprises at least8, at least 10, at least 12, at least 14, at least 16, or at least 18nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 190,215, 222, 223, 226, 246, and 254. In certain embodiments, the modifiedoligonucleotide comprises a nucleobase sequence selected from SEQ IDNOs: 34, 190, 215, 222, 223, 226, 246, and 254. In certain embodiments,the modified oligonucleotide consists of a nucleobase sequence selectedfrom SEQ ID NOs: 34, 190, 215, 222, 223, 226, 246, and 254. Saidmodified oligonucleotides may achieve at least 90% inhibition of humanmRNA levels as determined using an RT-PCR assay method, optionally inHepG2 cells (e.g. as described in Example 30).

In certain embodiments, the compound consists of a single-strandedmodified oligonucleotide.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is 100% complementary to a nucleobase sequence of SEQ IDNO: 1 or SEQ ID NO: 2 or SEQ ID NO: 6 or SEQ ID NO: 274.

In certain embodiments, the compound has at least one modifiedinternucleoside linkage. In certain embodiments, the internucleosidelinkage is a phosphorothioate internucleoside linkage.

In certain embodiments, the compound has at least one nucleosidecomprising a modified sugar. In certain embodiments, the at least onemodified sugar is a bicyclic sugar. In certain embodiments, the at leastone modified sugar comprises a 2′-O-methoxyethyl.

Embodiments of the present invention provide compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 18, or 20 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 15 to 241, SEQ ID NOs: 15 to 269, orSEQ ID NOs: 242 to 269, wherein at least one nucleoside comprises amodified sugar.

In certain embodiments, said at least one at least one modified sugar isa bicyclic sugar.

In certain embodiments, said at least one bicyclic sugar comprises a4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or 2.

In certain embodiments, said at least one bicyclic sugar comprises a4′-CH(CH3)-O-2′ bridge.

In certain embodiments, said at least one modified sugar comprises a2′-O-methoxyethyl group.

Embodiments of the present invention provide compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 18, or 20 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 15 to 241, SEQ ID NOs: 15 to 269, orSEQ ID NOs: 242 to 269, comprising at least one tetrahydropyran modifiednucleoside wherein a tetrahydropyran ring replaces the furanose ring.

In certain embodiments, said at least one tetrahydropyran modifiednucleoside has the structure:

In certain embodiments, the compound has at least one nucleosidecomprising a modified nucleobase. In certain embodiments, the modifiednucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   -   (i) a gap segment consisting of linked deoxynucleosides;    -   (ii) a 5′ wing segment consisting of linked nucleosides;    -   (iii) a 3′ wing segment consisting of linked nucleosides,        wherein the gap segment is positioned immediately adjacent to        and between the 5′ wing segment and the 3′ wing segment and        wherein each nucleoside of each wing segment comprises a        modified sugar. In some such embodiments, each cytosine in the        modified oligonucleotide is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   -   (i) a gap segment consisting of ten linked deoxynucleosides;    -   (ii) a 5′ wing segment consisting of five linked nucleosides;    -   (iii) a 3′ wing segment consisting of five linked nucleosides,        wherein the gap segment is positioned immediately adjacent to        and between the 5′ wing segment and the 3′ wing segment, wherein        each nucleoside of each wing segment comprises a        2′-O-methoxyethyl sugar; and wherein each internucleoside        linkage is a phosphorothioate linkage. In some such embodiments,        each cytosine in the modified oligonucleotide is a        5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   -   (i) a gap segment consisting of fourteen linked        deoxynucleosides;    -   (ii) a 5′ wing segment consisting of three linked nucleosides;    -   (iii) a 3′ wing segment consisting of three linked nucleosides,        wherein the gap segment is positioned immediately adjacent to        and between the 5′ wing segment and the 3′ wing segment, wherein        each nucleoside of each wing segment comprises a        2′-O-methoxyethyl sugar; and wherein each internucleoside        linkage is a phosphorothioate linkage. In some such embodiments,        each cytosine in the modified oligonucleotide is a        5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   -   (i) a gap segment consisting of thirteen linked        deoxynucleosides;    -   (ii) a 5′ wing segment consisting of two linked nucleosides;    -   (iii) a 3′ wing segment consisting of five linked nucleosides,        wherein the gap segment is positioned immediately adjacent to        and between the 5′ wing segment and the 3′ wing segment, wherein        each nucleoside of each wing segment comprises a        2′-O-methoxyethyl sugar; and wherein each internucleoside        linkage is a phosphorothioate linkage. In some such embodiments,        each cytosine in the modified oligonucleotide is a        5-methylcytosine.

Embodiments of the present invention provide a composition comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 12 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 15 to 241 or a salt thereof and apharmaceutically acceptable carrier or diluent.

Embodiments of the present invention provide a composition comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 12 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 15 to 269 or a salt thereof and apharmaceutically acceptable carrier or diluent.

Embodiments of the present invention provide a composition comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 12 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 241 to 269 or a salt thereof and apharmaceutically acceptable carrier or diluent.

Embodiments of the present invention provide methods comprisingadministering to an animal a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs: 15 to 241.

Embodiments of the present invention provide methods comprisingadministering to an animal a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs: 15 to 269.

Embodiments of the present invention provide methods comprisingadministering to an animal a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs: 241 to 269.

In certain embodiments, the animal is a human.

In certain embodiments, the administering prevents deep vein thrombosisor pulmonary embolism.

In certain embodiments, the compound is co-administered with any of thegroup selected from aspirin, clopidogrel, dipyridamole, heparin,lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX.

In certain embodiments, the compound is co-administered with any FactorXa inhibitor.

In certain embodiment, the Factor Xa inhibitor is any of Rivaroxaban,LY517717, YM150, apixaban, PRT054021, and DU-176b.

In certain embodiments, the compound is administered concomitantly withany of the group selected from aspirin, clopidogrel, dipyridamole,heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, andLOVENOX are administered concomitantly.

In certain embodiments, the administering is parenteral administration.In certain embodiments, the parenteral administration is any ofsubcutaneous or intravenous administration.

Embodiments of the present invention provide methods comprisingidentifying an animal at risk for developing thromboemboliccomplications and administering to the at risk animal a therapeuticallyeffective amount of a compound comprising a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is complementary to a Factor 11 nucleic acid.

In certain embodiments, the thromboembolic complication is deep veinthrombosis, pulmonary embolism, or a combination thereof.

Embodiments of the present invention provide methods comprisingidentifying an animal having a clotting disorder by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein the modified oligonucleotide is complementary to a Factor 11nucleic acid.

In certain embodiments, the compound is co-administered with any of thegroup selected from aspirin, clopidogrel, dipyridamole, heparin,lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX.

In certain embodiments, the compound is administered concomitantly withany of the group selected from aspirin, clopidogrel, dipyridamole,heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, andLOVENOX are administered concomitantly.

Embodiments of the present invention provide methods comprising reducingthe risk for thromboembolic complications in an animal by administeringto the animal a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the modified oligonucleotide is complementary to aFactor 11 nucleic acid.

Embodiments of the present invention provide methods comprising treatinga clotting disorder in an animal by administering to the animal atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein themodified oligonucleotide is complementary to a Factor 11 nucleic acid.

Embodiments of the present invention provide methods comprisinginhibiting Factor 11 expression in an animal by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein the modified oligonucleotide is complementary to a Factor 11nucleic acid.

In certain embodiments, the Factor 11 inhibition in the animal isreversed by administering an antidote to the modified oligonucleotide.

In certain embodiments, the antidote is an oligonucleotide complementaryto the modified oligonucleotide.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound may be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a Factor 11nucleic acid is 12 to 30 subunits in length. In other words, suchantisense compounds are from 12 to 30 linked subunits. In otherembodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18to 24, 19 to 22, or 20 linked subunits. In certain such embodiments, theantisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a rangedefined by any two of the above values. In some embodiments theantisense compound is an antisense oligonucleotide, and the linkedsubunits are nucleotides.

In certain embodiments antisense oligonucleotides targeted to a Factor11 nucleic acid may be shortened or truncated. For example, a singlesubunit may be deleted from the 5′ end (5′ truncation), or alternativelyfrom the 3′ end (3′ truncation). A shortened or truncated antisensecompound targeted to a Factor 11 nucleic acid may have two subunitsdeleted from the 5′ end, or alternatively may have two subunits deletedfrom the 3′ end, of the antisense compound. Alternatively, the deletednucleosides may be dispersed throughout the antisense compound, forexample, in an antisense compound having one nucleoside deleted from the5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisensecompound, the additional subunit may be located at the 5′ or 3′ end ofthe antisense compound. When two or more additional subunits arepresent, the added subunits may be adjacent to each other, for example,in an antisense compound having two subunits added to the 5′ end (5′addition), or alternatively to the 3′ end (3′ addition), of theantisense compound. Alternatively, the added subunits may be dispersedthroughout the antisense compound, for example, in an antisense compoundhaving one subunit added to the 5′ end and one subunit added to the 3′end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid have chemically modified subunits arranged in patterns, ormotifs, to confer to the antisense compounds properties such as enhancedthe inhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In certain embodiments, theregions of a gapmer are differentiated by the types of sugar moietiescomprising each distinct region. The types of sugar moieties that areused to differentiate the regions of a gapmer may in some embodimentsinclude β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modifiednucleosides (such 2′-modified nucleosides may include 2′-MOE, and2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (suchbicyclic sugar modified nucleosides may include those having a4′-(CH2)n-O-2′ bridge, where n=1 or n=2). Preferably, each distinctregion comprises uniform sugar moieties. The wing-gap-wing motif isfrequently described as “X-Y-Z”, where “X” represents the length of the5′ wing region, “Y” represents the length of the gap region, and “Z”represents the length of the 3′ wing region. As used herein, a gapmerdescribed as “X-Y-Z” has a configuration such that the gap segment ispositioned immediately adjacent each of the 5′ wing segment and the 3′wing segment. Thus, no intervening nucleotides exist between the 5′ wingsegment and gap segment, or the gap segment and the 3′ wing segment. Anyof the antisense compounds described herein can have a gapmer motif. Insome embodiments, X and Z are the same, in other embodiments they aredifferent. In a preferred embodiment, Y is between 8 and 15 nucleotides.X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmers of thepresent invention include, but are not limited to, for example 5-10-5,4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2,1-10-1, 2-8-2, 5-8-5, or 6-8-6.

In certain embodiments, the antisense compound has a “wingmer” motif,having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Zconfiguration as described above for the gapmer configuration. Thus,wingmer configurations of the present invention include, but are notlimited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3,2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8.

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid possess a 3-14-3 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid possess a 2-13-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid possess a 5-8-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid possess a 6-8-6 gapmer motif.

In certain embodiments, an antisense compound targeted to a Factor 11nucleic acid has a gap-widened motif.

In certain embodiments, a gap-widened antisense oligonucleotide targetedto a Factor 11 nucleic acid has a gap segment of fourteen2′-deoxyribonucleotides positioned immediately adjacent to and betweenwing segments of three chemically modified nucleosides. In certainembodiments, the chemical modification comprises a 2′-sugarmodification. In another embodiment, the chemical modification comprisesa 2′-MOE sugar modification.

In certain embodiments, a gap-widened antisense oligonucleotide targetedto a Factor 11 nucleic acid has a gap segment of thirteen2′-deoxyribonucleotides positioned immediately adjacent to and between a5′ wing segment of two chemically modified nucleosides and a 3′ wingsegment of five chemically modified nucleosides. In certain embodiments,the chemical modification comprises a 2′-sugar modification. In anotherembodiment, the chemical modification comprises a 2′-MOE sugarmodification.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode Factor 11 include, without limitation,the following: GENBANK Accession No. NM_000128.3, first deposited withGENBANK on Mar. 24, 1999 incorporated herein as SEQ ID NO: 1;NT_022792.17, truncated from 19598000 to 19624000, first deposited withGENBANK on Nov. 29, 2000, and incorporated herein as SEQ ID NO: 2;GENBANK Accession No. NM_028066.1, first deposited with GENBANK on Jun.2, 2002, incorporated herein as SEQ ID NO: 6; and exons 1-15 GENBANKAccession No. NW_001118167.1, first deposited with GENBANK on Mar. 28,2006, incorporated herein as SEQ ID NO: 274.

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor Factor 11 can be obtained by accession number from sequencedatabases such as NCBI and such information is incorporated herein byreference. In certain embodiments, a target region may encompass thesequence from a 5′ target site of one target segment within the targetregion to a 3′ target site of another target segment within the sametarget region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceeding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inFactor 11 mRNA levels are indicative of inhibition of Factor 11expression. Reductions in levels of a Factor 11 protein are alsoindicative of inhibition of target mRNA expression. Further, phenotypicchanges are indicative of inhibition of Factor 11 expression. Forexample, a prolonged aPTT time can be indicative of inhibition of Factor11 expression. In another example, prolonged aPTT time in conjunctionwith a normal PT time can be indicative of inhibition of Factor 11expression. In another example, a decreased quantity of Platelet Factor4 (PF-4) can be indicative of inhibition of Factor 11 expression. Inanother example, reduced formation of thrombus or increased time forthrombus formation can be indicative of inhibition of Factor 11expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a Factor 11 nucleic acid. The most common mechanismof hybridization involves hydrogen bonding (e.g., Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the antisense compounds provided herein are specificallyhybridizable with a Factor 11 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a Factor 11nucleic acid).

Non-complementary nucleobases between an antisense compound and a Factor11 nucleic acid may be tolerated provided that the antisense compoundremains able to specifically hybridize to a target nucleic acid.Moreover, an antisense compound may hybridize over one or more segmentsof a Factor 11 nucleic acid such that intervening or adjacent segmentsare not involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a Factor 11 nucleic acid, a target region, targetsegment, or specified portion thereof. Percent complementarity of anantisense compound with a target nucleic acid can be determined usingroutine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, antisense compound may be fully complementary to a Factor11 nucleic acid, or a target region, or a target segment or targetsequence thereof. As used herein, “fully complementary” means eachnucleobase of an antisense compound is capable of precise base pairingwith the corresponding nucleobases of a target nucleic acid. Forexample, a 20 nucleobase antisense compound is fully complementary to atarget sequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e. linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no morethan 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a Factor 11 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a Factor 11 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 12 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least a 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a targetsegment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is comparedto an equal length portion of the target nucleic acid. In certainembodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleobase portion is compared to an equal lengthportion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide iscompared to an equal length portion of the target nucleic acid. Incertain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equallength portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages arephosphorothioate linkages. In certain embodiments, each internucleosidelinkage of an antisense compound is a phosphorothioate internucleosidelinkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprise achemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C1-C12alkyl or a protecting group) and combinations thereof. Examples ofchemically modified sugars include 2′-F-5′-methyl substituted nucleoside(see PCT International Application WO 2008/101157 Published on Aug. 21,2008 for other disclosed 5′,2′-bis substituted nucleosides) orreplacement of the ribosyl ring oxygen atom with S with furthersubstitution at the 2′-position (see published U.S. Patent ApplicationUS2005-0130923, published on Jun. 16, 2005) or alternatively5′-substitution of a BNA (see PCT International Application WO2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted withfor example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH3 and 2′-O(CH2)2OCH3 substituent groups. The substituent atthe 2′ position can also be selected from allyl, amino, azido, thio,O-allyl, O—C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-O—N(Rm)(Rn), andO-CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H orsubstituted or unsubstituted C1-C10 alkyl.

Examples of bicyclic nucleic acids (BNAs) include without limitationnucleosides comprising a bridge between the 4′ and the 2′ ribosyl ringatoms. In certain embodiments, antisense compounds provided hereininclude one or more BNA nucleosides wherein the bridge comprises one ofthe formulas: 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)-O-2′ (LNA);4′-(CH2)2-O-2′ (ENA); 4′-C(CH3)2-O-2′ (see PCT/US2008/068922);4′-CH(CH3)¬-O-2′ and 4′-C¬H(CH2OCH3)¬-O-2′ (see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-CH2-N(OCH3)-2′ (see PCT/US2008/064591);4′-CH2-O—N(CH3)-2′ (see published U.S. Patent ApplicationUS2004-0171570, published Sep. 2, 2004); 4′-CH2-N(R)—O-2′ (see U.S. Pat.No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2-C(CH3)-2′ and4′-CH2-C¬(=CH2)-2′ (see PCT/US2008/066154); and wherein R is,independently, H, C1-C12 alkyl, or a protecting group. Each of theforegoing BNAs include various stereochemical sugar configurationsincluding for example α-L-ribofuranose and β-D-ribofuranose (see PCTinternational application PCT/DK98/00393, published on Mar. 25, 1999 asWO 99/14226).

In certain embodiments, nucleosides are modified by replacement of theribosyl ring with a sugar surrogate. Such modification includes withoutlimitation, replacement of the ribosyl ring with a surrogate ring system(sometimes referred to as DNA analogs) such as a morpholino ring, acyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such asone having one of the formula:

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknow in the art that can be used to modify nucleosides for incorporationinto antisense compounds (see for example review article: Leumann, J. C,Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Such ring systems can undergo various additional substitutions toenhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds targeted to a MMP-13 nucleicacid comprise one or more nucleotides having modified sugar moieties. Incertain embodiments, the modified sugar moiety is 2′-MOE. In certainembodiments, the 2′-MOE modified nucleotides are arranged in a gapmermotif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases.

Both natural and modified nucleobases are capable of participating inhydrogen bonding. Such nucleobase modifications may impart nucleasestability, binding affinity or some other beneficial biological propertyto antisense compounds. Modified nucleobases include synthetic andnatural nucleobases such as, for example, 5-methylcytosine (5-me-C).Certain nucleobase substitutions, including 5-methylcytosinesubstitutions, are particularly useful for increasing the bindingaffinity of an antisense compound for a target nucleic acid. Forexample, 5-methylcytosine substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press,Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a Factor 11nucleic acid comprise one or more modified nucleobases. In certainembodiments, gap-widened antisense oligonucleotides targeted to a Factor11 nucleic acid comprise one or more modified nucleobases.

In certain embodiments, the modified nucleobase is 5-methylcytosine. Incertain embodiments, each cytosine is a 5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

An antisense compound targeted to a Factor 11 nucleic acid can beutilized in pharmaceutical compositions by combining the antisensecompound with a suitable pharmaceutically acceptable diluent or carrier.A pharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to a Factor 11 nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof Factor 11 nucleic acids can be tested in vitro in a variety of celltypes. Cell types used for such analyses are available from commercialvendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio,Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville,Md.) and are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, and primary hepatocytes.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a LIPOFECTAMINEconcentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE. Antisense oligonucleotides are used at higherconcentrations ranging from 625 to 20,000 nM when transfected usingelectroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOLReagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a Factor 11 nucleic acid can beassayed in a variety of ways known in the art. For example, targetnucleic acid levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or quantitative real-timePCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Methods of quantitative real-time PCR arewell known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000instrument (PE Applied Biosystems) is used to measure RIBOGREENfluorescence.

Probes and primers are designed to hybridize to a Factor 11 nucleicacid. Methods for designing real-time PCR probes and primers are wellknown in the art, and may include the use of software such as PRIMEREXPRESS Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of Factor 11 nucleic acids can be assessed bymeasuring Factor 11 protein levels. Protein levels of Factor 11 can beevaluated or quantitated in a variety of ways well known in the art,such as immunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art. Antibodies useful for the detection of mouse,rat, monkey, and human Factor 11 are commercially available.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of Factor 11and produce phenotypic changes, such as, prolonged aPTT, prolonged aPTTtime in conjunction with a normal PT, decreased quantity of PlateletFactor 4 (PF-4), and reduced formation of thrombus or increased time forthrombus formation. Testing may be performed in normal animals, or inexperimental disease models. For administration to animals, antisenseoligonucleotides are formulated in a pharmaceutically acceptablediluent, such as phosphate-buffered saline. Administration includesparenteral routes of administration, such as intraperitoneal,intravenous, and subcutaneous. Calculation of antisense oligonucleotidedosage and dosing frequency is within the abilities of those skilled inthe art, and depends upon factors such as route of administration andanimal body weight. Following a period of treatment with antisenseoligonucleotides, RNA is isolated from liver tissue and changes inFactor 11 nucleic acid expression are measured. Changes in Factor 11protein levels are also measured using a thrombin generation assay. Inaddition, effects on clot times, e.g. PT and aPTT, are determined usingplasma from treated animals.

Tolerability

In certain embodiments, the compounds provided herein display minimalside effects. Side effects include responses to the administration ofthe antisense compound that are typically unrelated to the targeting offactor 11, such as an inflammatory response in the animal. In certainembodiments compounds are well tolerated by the animal. Increasedtolerability can depend on a number of factors, including, but notlimited to, the nucleotide sequence of the antisense compound, chemicalmodifications to the nucleotides, the particular motif of unmodified andmodified nucleosides in the antisense compound, or combinations thereof.Tolerability may be determined by a number of factors. Such factorsinclude body weight, organ weight, liver function, kidney function,platelet count, white blood cell count.

In certain embodiments, the compounds provided herein demonstrateminimal effect on organ weight. In certain embodiments, the compoundsdemonstrate less than a 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-foldor no significant increase in spleen and/or liver weight.

In certain embodiments, the compounds provided herein demonstrateminimal effect on liver function. Factors for the evaluation of liverfunction include ALT levels, AST levels, plasma bilirubin levels andplasma albumin levels. In certain embodiments the compounds providedherein demonstrate less than a 7-fold, less than a 6-fold, less than a5-fold, less than a 4-fold, less than a 3-fold or less than a 2-fold orno significant increase in ALT or AST. In certain embodiments thecompounds provided herein demonstrate less than a 3-fold, less than a2-fold or no significant increase in plasma bilirubin levels.

In certain embodiments, the compounds provided herein demonstrateminimal effect on kidney function. In certain embodiments, the compoundsprovided herein demonstrate less than a 3-fold, less than a 2-fold, orno significant increase in plasma concentrations of blood urea nitrogen(BUN). In certain embodiments, the compounds provided herein demonstrateless than a 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or no significantincrease in the ratio of urine protein to creatinine.

In certain embodiments, the compounds provided herein demonstrateminimal effect on hematological factors. In certain embodiments, thecompounds provided herein demonstrate less than a 60%, 50%, 40%, 30%,20%, 10% or 5% decrease in platelet count. In certain embodiments, thecompounds provided herein demonstrate less than a 4-fold, less than a3-fold, less than a 2-fold or no significant increase in monocyte count.

In certain embodiments compounds further display favorablepharmacokinetics. In certain embodiments, antisense compounds exhibitrelatively high half-lives in relevant biological fluids or tissues.

In certain embodiments, compounds or compositions further displayfavorable viscosity. In certain embodiments, the viscosity of thecompound or composition is no more than 40 cP at a concentration of165-185 mg/mL.

In other embodiments, the compounds display combinations of thecharacteristics above and reduce factor 11 mRNA expression in an animalmodel with high efficiency.

Certain Indications

In certain embodiments, the invention provides methods of treating anindividual comprising administering one or more pharmaceuticalcompositions of the present invention. In certain embodiments, theindividual has a thromboembolic complication. In certain embodiments,the individual is at risk for a blood clotting disorder, including, butnot limited to, infarct, thrombosis, embolism, thromboembolism such asdeep vein thrombosis, pulmonary embolism, myocardial infarction, andstroke. This includes individuals with an acquired problem, disease, ordisorder that leads to a risk of thrombosis, for example, surgery,cancer, immobility, sepsis, atherosclerosis atrial fibrillation, as wellas genetic predisposition, for example, antiphospholipid syndrome andthe autosomal dominant condition, Factor V Leiden. In certainembodiments, the individual has been identified as in need ofanticoagulation therapy. Examples of such individuals include, but arenot limited to, those undergoing major orthopedic surgery (e.g.,hip/knee replacement or hip fracture surgery) and patients in need ofchronic treatment, such as those suffering from arterial fibrillation toprevent stroke. In certain embodiments the invention provides methodsfor prophylactically reducing Factor 11 expression in an individual.Certain embodiments include treating an individual in need thereof byadministering to an individual a therapeutically effective amount of anantisense compound targeted to a Factor 11 nucleic acid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted to a Factor 11 nucleic acid isaccompanied by monitoring of Factor 11 levels in the serum of anindividual, to determine an individual's response to administration ofthe antisense compound. An individual's response to administration ofthe antisense compound is used by a physician to determine the amountand duration of therapeutic intervention.

In certain embodiments, administration of an antisense compound targetedto a Factor 11 nucleic acid results in reduction of Factor 11 expressionby at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 99%, or a range defined by any two of these values. In certainembodiments, administration of an antisense compound targeted to aFactor 11 nucleic acid results in a change in a measure of bloodclotting as measured by a standard test, for example, but not limitedto, activated partial thromboplastin time (aPTT) test, prothrombin time(PT) test, thrombin time (TCT), bleeding time, or D-dimer. In certainembodiments, administration of a Factor 11 antisense compound increasesthe measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues. In some embodiments, administration of a Factor 11 antisensecompound decreases the measure by at least 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by anytwo of these values.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to Factor 11 are used for the preparation ofa medicament for treating a patient suffering or susceptible to athromboembolic complication.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositions of thepresent invention are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease, disorder,or condition as the one or more pharmaceutical compositions of thepresent invention. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat a different disease,disorder, or condition as the one or more pharmaceutical compositions ofthe present invention. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat an undesired side effect ofone or more pharmaceutical compositions of the present invention.

In certain embodiments, one or more pharmaceutical compositions of thepresent invention are co-administered with another pharmaceutical agentto treat an undesired effect of that other pharmaceutical agent. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention are co-administered with another pharmaceutical agentto produce a combinational effect. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to produce a synergistic effect.

In certain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at the same time. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at different times. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areprepared together in a single formulation. In certain embodiments, oneor more pharmaceutical compositions of the present invention and one ormore other pharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include anticoagulant or antiplatelet agents. In certainembodiments, pharmaceutical agents that may be co-administered with apharmaceutical composition of the present invention includeNSAID/Cyclooxygenase inhibitors, such as, aspirin. In certainembodiments, pharmaceutical agents that may be co-administered with apharmaceutical composition of the present invention include adenosinediphosphate (ADP) receptor inhibitors, such as, clopidogrel (PLAVIX) andticlopidine (TICLID). In certain embodiments, pharmaceutical agents thatmay be co-administered with a pharmaceutical composition of the presentinvention include phosphodiesterase inhibitors, such as, cilostazol(PLETAL). In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include, glycoprotein IIB/IIIA inhibitors, such as, abciximab(REOPRO), eptifibatide (INTEGRILIN), tirofiban (AGGRASTAT), anddefibrotide. In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include, adenosine reuptake inhibitors, such as, todipyridamole (PERSANTINE). In certain embodiments, pharmaceutical agentsthat may be co-administered with a pharmaceutical composition of thepresent invention include, but are not limited to warfarin (and relatedcoumarins), heparin, direct thrombin inhibitors (such as lepirudin,bivalirudin), apixaban, LOVENOX, and small molecular compounds thatinterfere directly with the enzymatic action of particular coagulationfactors (e.g. rivaroxaban, which interferes with Factor Xa). In certainembodiments, pharmaceutical agents that may be co-administered with aFactor 11 specific inhibitor of the present invention include, but arenot limited to, an additional Factor 11 inhibitor. In certainembodiments, the anticoagulant or antiplatelet agent is administeredprior to administration of a pharmaceutical composition of the presentinvention. In certain embodiments, the anticoagulant or antiplateletagent is administered following administration of a pharmaceuticalcomposition of the present invention. In certain embodiments theanticoagulant or antiplatelet agent is administered at the same time asa pharmaceutical composition of the present invention. In certainembodiments the dose of a co-administered anticoagulant or antiplateletagent is the same as the dose that would be administered if theanticoagulant or antiplatelet agent was administered alone. In certainembodiments the dose of a co-administered anticoagulant or antiplateletagent is lower than the dose that would be administered if theanticoagulant or antiplatelet agent was administered alone. In certainembodiments the dose of a co-administered anticoagulant or antiplateletagent is greater than the dose that would be administered if theanticoagulant or antiplatelet agent was administered alone.

In certain embodiments, the co-administration of a second compoundenhances the anticoagulant effect of a first compound, such thatco-administration of the compounds results in an anticoagulant effectthat is greater than the effect of administering the first compoundalone. In other embodiments, the co-administration results inanticoagulant effects that are additive of the effects of the compoundswhen administered alone. In certain embodiments, the co-administrationresults in anticoagulant effects that are supra-additive of the effectsof the compounds when administered alone. In certain embodiments, theco-administration of a second compound increases antithrombotic activitywithout increased bleeding risk. In certain embodiments, the firstcompound is an antisense compound. In certain embodiments, the secondcompound is an antisense compound.

In certain embodiments, an antidote is administered anytime after theadministration of a Factor 11 specific inhibitor. In certainembodiments, an antidote is administered anytime after theadministration of an antisense oligonucleotide targeting Factor 11. Incertain embodiments, the antidote is administered minutes, hours, days,weeks, or months after the administration of an antisense compoundtargeting Factor 11. In certain embodiments, the antidote is acomplementary (e.g. the sense strand) to the antisense compoundtargeting Factor 11. In certain embodiments, the antidote is a Factor 7,Factor 7a, Factor 11, or Factor 11a protein. In certain embodiments, theFactor 7, Factor 7a, Factor 11, or Factor 11a protein is a human Factor7, human Factor 7a, human Factor 11, or human Factor 11a protein. Incertain embodiments, the Factor 7 protein is NOVOSEVEN.

Certain Co-Administered Antiplatelet Therapies

In certain embodiments, Factor 11 inhibitors are combined withantiplatelet therapies. In certain embodiments, administration of aFactor 11 inhibitor in combination with an antiplatelet therapy resultsin little to no appreciable or detectable increase in risk of bleedingas compared to antiplatelet therapy alone. In certain embodiments, therisk profile or risk indications are unchanged over antiplatelet therapyalone.

The combination of antiplatelet and anticoagulant therapy is used inclinical practice most frequently in patients diagnosed with, forexample, thromboembolism, atrial fibrillation, a heart valve disorder,valvular heart disease, stroke, CAD, and in patients having a mechanicalvalve. The benefit of dual therapy relates to the probable additiveeffect of suppressing both platelet and coagulation factor activities.The risk of dual therapy is the potential for increased bleeding (Dowd,M. Plenary Sessions/Thrombosis Research 123 (2008)).

Prior combinations of antiplatelet and anticoagulant therapy have beenshown to increase the risk of bleeding compared with anticoagulant orantiplatelet therapy alone. Such combinations include, FXa inhibitors(e.g., apixiban and rivaroxaban) with ADP receptor/P2Y12 inhibitors(Thienopyridines such as clopidogrel—also known as PLAVIX) and NSAIDs(e.g., aspirin and naproxen) (Kubitza, D. et al., Br. J. Clin.Pharmacol. 63:4 (2006); Wong, P. C. et al. Journal of Thrombosis andHaemostasis 6 (2008); FDA Advisory Committee Briefing Document for NewDrug Application 22-406 (2009)). For example, Wong reports that additionof certain doses of apixaban to aspirin and to aspirin plus clopidogrelproduced a significant increase in bleeding time compared with aspirinalone and asprin plus clopidogrel. Kubitza reports that the combinationadministration of rivaroxaban and naproxen significantly increasedbleeding time over naproxen alone.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Antisense Inhibition of Human Factor 11 in HepG2 Cells

Antisense oligonucleotides targeted to a Factor 11 nucleic acid weretested for their effects on Factor 11 mRNA in vitro. Cultured HepG2cells at a density of 10,000 cells per well were transfected usinglipofectin reagent with 75 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and Factor 11 mRNA levels were measured by quantitative real timePCR. Factor 11 mRNA levels were adjusted according to total RNA content,as measured by RIBOGREEN. Results are presented as percent inhibition ofFactor 11, relative to untreated control cells.

The chimeric antisense oligonucleotides in Tables 1 and 2 were designedas 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, whereinthe central gap segment is comprised of 10 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprising5 nucleotides each. Each nucleotide in the 5′ wing segment and eachnucleotide in the 3′ wing segment has a 2′-MOE modification. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P=S) linkages. All cytidine residues throughout each gapmer are5-methylcytidines. “Target start site” indicates the 5′-most nucleotideto which the gapmer is targeted. “Target stop site” indicates the3′-most nucleotide to which the gapmer is targeted. Each gapmer listedin Table 1 is targeted to SEQ TD NO: 1 (GENBANK Accession No.NM_000128.3) and each gapmer listed in Table 2 is targeted to SEQ TD NO:2 (GENBANK Accession No. NT_022792.17, truncated from 19598000 to19624000).

TABLE 1 Inhibition of human Factor 11 mRNA levels bychimeric antisense oligonucleotides having5-10-5 MOE wings and deoxy gap targeted to SEQ ID NO: 1 Tar- Tar- getget % SEQ Oligo Start Stop inhibi- ID ID Site Site Sequence tion NO412187 38 57 TTCAAACAAGTGACATACAC 21 15 412188 96 115TGAGAGAATTGCTTGCTTTC 21 16 412189 106 125 AAATATACCTTGAGAGAATT 8 17412190 116 135 AGTATGTCAGAAATATACCT 24 18 412191 126 145TTAAAATCTTAGTATGTCAG 14 19 412192 146 165 CAGCATATTTGTGAAAGTCG 44 20412193 222 241 TGTGTAGGAAATGGTCACTT 38 21 412194 286 305TGCAATTCTTAATAAGGGTG 80 22 412195 321 340 AAATCATCCTGAAAAGACCT 22 23412196 331 350 TGATATAAGAAAATCATCCT 25 24 412197 376 395ACACATTCACCAGAAACTGA 45 25 412198 550 569 TTCAGGACACAAGTAAACCA 21 26412199 583 602 TTCACTCTTGGCAGTGTTTC 66 27 412200 612 631AAGAATACCCAGAAATCGCT 59 28 412201 622 641 CATTGCTTGAAAGAATACCC 66 29412202 632 651 TTGGTGTGAGCATTGCTTGA 65 30 412203 656 675AATGTCTTTGTTGCAAGCGC 91 31 412204 676 695 TTCATGTCTAGGTCCACATA 74 32412205 686 705 GTTTATGCCCTTCATGTCTA 69 33 412206 738 757CCGTGCATCTTTCTTGGCAT 87 34 412207 764 783 CGTGAAAAAGTGGCAGTGGA 64 35412208 811 830 AGACAAATGTTACGATGCTC 73 36 412209 821 840GTGCTTCAGTAGACAAATGT 91 37 412210 896 915 TGCACAGGATTTCAGTGAAA 73 38412211 906 925 GATTAGAAAGTGCACAGGAT 64 39 412212 1018 1037CCGGGATGATGAGTGCAGAT 88 40 412213 1028 1047 AAACAAGCAACCGGGATGAT 71 4412214 1048 1067 TCCTGGGAAAAGAAGGTAAA 58 42 412215 1062 1081ATTCTTTGGGCCATTCCTGG 81 43 412216 1077 1096 AAAGATTTCTTTGAGATTCT 43 44412217 1105 1124 AATCCACTCTCAGATGTTTT 47 45 412218 1146 1165AACCAGAAAGAGCTTTGCTC 27 46 412219 1188 1207 GGCAGAACACTGGGATGCTG 56 47412220 1204 1223 TGGTAAAATGAAGAATGGCA 58 48 412221 1214 1233ATCAGTGTCATGGTAAAATG 48 49 412222 1241 1263 AACAATATCCAGTTCTTCTC 5 50412223 1275 1294 ACAGTTTCTGGCAGGCCTCG 84 51 412224 1285 1304GCATTGGTGCACAGTTTCTG 87 52 412225 1295 1314 GCAGCGGACGGCATTGGTGC 86 53412226 1371 1390 TTGAAGAAAGCTTTAAGTAA 17 54 412227 1391 1410AGTATTTTAGTTGGAGATCC 75 55 412228 1425 1444 ATGTGTATCCAGAGATGCCT 71 56412229 1456 1475 GTACACTCATTATCCATTTT 64 57 412230 1466 1485GATTTTGGTGGTACACTCAT 52 58 412231 1476 1495 TCCTGGGCTTGATTTTGGTG 74 59412232 1513 1532 GGCCACTCACCACGAACAGA 80 60 412233 1555 1574TGTCTCTGAGTGGGTGAGGT 64 61 412234 1583 1602 GTTTCCAATGATGGAGCCTC 60 62412235 1593 1612 ATATCCACTGGTTTCCAATG 57 63 412236 1618 1637CCATAGAAACAGTGAGCGGC 72 64 412237 1628 1647 TGACTCTACCCCATAGAAAC 48 65412238 1642 1661 CGCAAAATCTTAGGTGACTC 71 66 412239 1673 1692TTCAGATTGATTTAAAATGC 43 67 412240 1705 1724 TGAACCCCAAAGAAAGATGT 32 68412241 1715 1734 TATTATTTCTTGAACCCCAA 41 69 412242 1765 1784AACAAGGCAATATCATACCC 49 70 412243 1775 1794 TTCCAGTTTCAACAAGGCAA 70 71412244 1822 1841 GAAGGCAGGCATATGGGTCG 53 72 412245 1936 1955GTCACTAAGGGTATCTTGGC 75 73 412246 1992 2011 AGATCATCTTATGGGTTATT 68 74412247 2002 2021 TAGCCGGCACAGATCATCTT 75 75 412248 2082 2101CCAGATGCCAGACCTCATTG 53 76 412249 2195 2214 CATTCACACTGCTTGAGTTT 55 77412250 2268 2287 TGGCACAGTGAACTCAACAC 63 78 412251 2326 2345CTAGCATTTTCTTACAAACA 58 79 412252 2450 2469 TTATGGTAATTCTTGGACTC 39 80412253 2460 2479 AAATATTGCCTTATGGTAAT 20 81 412254 2485 2504TATCTGCCTATATAGTAATC 16 82 412255 2510 2529 GCCACTACTTGGTTATTTTC 38 83412256 2564 2583 AACAAATCTATTTATGGTGG 39 84 412257 2622 2641CTGCAAAATGGTGAAGACTG 57 85 412258 2632 2651 GTGTAGATTCCTGCAAAATG 44 86412259 2882 2901 TTTTCAGGAAAGTGTATCTT 37 87 412260 2892 2911CACAAATCATTTTTCAGGAA 27 88 412261 2925 2944 TCCCAAGATATTTTAAATAA 3 89412262 3168 3187 AATGAGATAAATATTTGCAC 34 90 412263 3224 3243TGAAAGCTATGTGGTGACAA 33 91 412264 3259 3278 CACACTTGATGAATTGTATA 27 92413460 101 120 TACCTTGAGAGAATTGCTTG 40 93 413461 111 130GTCAGAAATATACCTTGAGA 39 94 413462 121 140 ATCTTAGTATGTCAGAAATA 12 95413463 381 400 GAGTCACACATTCACCAGAA 74 96 413464 627 646GTGAGCATTGCTTGAAAGAA 42 97 413465 637 656 CTTATTTGGTGTGAGCATTG 80 98413466 661 680 ACATAAATGTCTTTGTTGCA 79 99 413467 666 685GGTCCACATAAATGTCTTTG 91 100 413468 671 690 GTCTAGGTCCACATAAATGT 84 101413469 681 700 TGCCCTTCATGTCTAGGTCC 84 102 413470 692 711GTTATAGTTTATGCCCTTCA 72 103 413471 816 835 TCAGTAGACAAATGTTACGA 67 104413472 826 845 TGGGTGTGCTTCAGTAGACA 99 105 413473 911 930AGCCAGATTAGAAAGTGCAC 80 106 413474 1023 1042 AGCAACCGGGATGATGAGTG 84 107413475 1053 1072 GCCATTCCTGGGAAAAGAAG 80 108 413476 1067 1086TTGAGATTCTTTGGGCCATT 88 109 413477 1151 1170 ACTGAAACCAGAAAGAGCTT 54 110413478 1193 1212 AGAATGGCAGAACACTGGGA 53 111 413479 1209 1228TGTCATGGTAAAATGAAGAA 40 112 413480 1219 1238 AAGAAATCAGTGTCATGGTA 71 113413481 1280 1299 GGTGCACAGTTTCTGGCAGG 86 114 413482 1290 1309GGACGGCATTGGTGCACAGT 85 115 413483 1300 1319 AACTGGCAGCGGACGGCATT 78 116413484 1430 1449 CCTTAATGTGTATCCAGAGA 74 117 413485 1461 1480TGGTGGTACACTCATTATCC 68 118 413486 1471 1490 GGCTTGATTTTGGTGGTACA 83 119413487 1481 1500 AACGATCCTGGGCTTGATTT 57 120 413488 1560 1579ACAGGTGTCTCTGAGTGGGT 49 121 413489 1588 1607 CACTGGTTTCCAATGATGGA 68 122413490 1623 1642 CTACCCCATAGAAACAGTGA 57 123 413491 1633 1652TTAGGTGACTCTACCCCATA 73 124 413492 1647 1666 AGACACGCAAAATCTTAGGT 68 125413493 1710 1729 TTTCTTGAACCCCAAAGAAA 65 126 413494 1780 1799GTGGTTTCCAGTTTCAACAA 70 127 413495 1921 1940 TTGGCTTTCTGGAGAGTATT 58 128413496 1997 2016 GGCACAGATCATCTTATGGG 72 129 413497 2627 2646GATTCCTGCAAAATGGTGAA 39 130 413498 2637 2656 GCAGAGTGTAGATTCCTGCA 60 131413499 2887 2906 ATCATTTTTCAGGAAAGTGT 52 132

TABLE 2 Inhibition of human Factor 11 mRNA levels bychimeric antisense oligonucleotides having5-10-5 MOE wings and deoxy gap targeted to SEQ ID NO: 2 Tar- Tar- getget % SEQ Oligo Start Stop inhibi- ID ID Site Site Sequence tion NO413500 1658 1677 GTGAGACAAATCAAGACTTC 15 133 413501 2159 2178TTAGTTTACTGACACTAAGA 23 134 413502 2593 2612 CTGCTTTATGAAAAACCAAC 22 135413503 3325 3344 ATACCTAGTACAATGTAAAT 29 136 413504 3548 3567GGCTTGTGTGTGGTCAATAT 54 137 413505 5054 5073 TGGGAAAGCTTTCAATATTC 57 138413506 6474 6493 ATGGAATTGTGCTTATGAGT 57 139 413507 7590 7609TTTCAAGCTCAGGATGGGAA 55 140 413508 7905 7924 GTTGGTAAAATGCAACCAAA 64 141413509 8163 8182 TCAGGACACAAGTAAACCTG 66 142 413510 9197 9216TGCAAGCTGGAAATAAAAGC 17 143 413511 9621 9640 TGCCAATTTAAAAGTGTAGC 43 144413512 9800 9819 ATATTTCAAAATCCAGTATG 39 145 413513 9919 9938TTCTGAATATACAAATTAAT 27 146 413514 9951 9970 TTTACTATGAAAATCTAAAT 5 147413515 11049 11068 GGTATCCTGAGTGAGATCTA 36 148 413516 11269 11288CCAGCTATCAGGAAAATTCC 50 149 413517 12165 12184 AAAGCTATTGGAGACTCAGA 51150 413518 12584 12603 ATGGAATCTCTTCATTTCAT 49 151 413519 12728 12747ATGGAGACATTCATTTCCAC 59 152 413520 13284 13303 GCTCTGAGAGTTCCAATTCA 52153 413521 14504 14523 CTGGGAAGGTGAATTTTTAG 62 154 413522 14771 14790TCAAGAGTCTTCATGCTACC 42 155 413523 15206 15225 TCAGTTTACCTGGGATGCTG 61156 413524 15670 15689 GACATTATACTCACCATTAT 7 157 413525 15905 15924GTATAAATGTGTCAAATTAA 43 158 413526 16482 16501 GTAAAGTTTTACCTTAACCT 47159 413527 17298 17317 CCATAATGAAGAAGGAAGGG 52 160 413528 17757 17776TTAAGTTACATTGTAGACCA 48 161 413529 18204 18223 TGTGTGGGTCCTGAAATTCT 52162 413530 18981 19000 ATCTTGTAATTACACACCCC 27 163 413531 19174 19193GTACACTCTGCAACAGAAGC 47 164 413532 19604 19623 AGGGAATAACATGAAGGCCC 32165 413533 20936 20955 ATCCAGTTCACCATTGGAGA 48 166 413534 21441 21460TTTTCCAGAAGAGACTCTTC 31 167 413535 21785 21804 GTCACATTTAAAATTTCCAA 41168 413536 23422 23441 TTAATATACTGCAGAGAACC 37 169 413537 25893 25912AGAAATATCCCCAGACAGAG 16 170

Example 2: Dose-Dependent Antisense Inhibition of Human Factor 11 inHepG2 Cells

Twelve gapmers, exhibiting over 84 percent or greater in vitroinhibition of human Factor 11, were tested at various doses in HepG2cells. Cells were plated at a density of 10,000 cells per well andtransfected using lipofectin reagent with 9.375 nM, 18.75 nM, 37.5 nM,75 nM, and 150 nM concentrations of antisense oligonucleotide, asspecified in Table 3. After a treatment period of approximately 16hours, RNA was isolated from the cells and Factor 11 mRNA levels weremeasured by quantitative real-time PCR. Human Factor 11 primer probe setRTS 2966 (forward sequence: CAGCCTGGAGCATCGTAACA, incorporated herein asSEQ ID NO: 3; reverse sequence: TTTATCGAGCTTCGTTATTCTGGTT, incorporatedherein as SEQ ID NO: 4; probe sequence: TTGTCTACTGAAGCACACCCAAACAGGGAX,incorporated herein as SEQ ID NO: 5) was used to measure mRNA levels.Factor 11 mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN. Results are presented as percent inhibition ofFactor 11, relative to untreated control cells. As illustrated in Table3, Factor 11 mRNA levels were reduced in a dose-dependent manner inantisense oligonucleotide treated cells.

TABLE 3 Dose-dependent antisense inhibition of human Factor 11 in HepG2cells 9.375 18.75 37.5 75 150 IC₅₀ SEQ ID nM nM nM nM nM (nM) No. 41220329 15 61 77 82 33 31 412206 28 44 68 80 89 22 34 412212 28 45 59 73 8825 40 412223 33 48 62 76 81 21 51 412224 24 45 57 70 81 28 52 412225 3242 65 78 73 23 53 413467 2 35 49 61 47 43 100 413468 14 34 56 78 75 35101 413469 24 33 53 70 84 33 102 413476 26 44 64 73 82 25 109 413481 2238 56 67 83 32 114 413482 26 39 59 74 82 28 115

Example 3: Antisense Inhibition of Human Factor 11 in HepG2 Cells byOligonucleotides Designed by Microwalk

Additional gapmers were designed based on the gapmers presented in Table3. These gapmers were designed by creating gapmers shifted slightlyupstream and downstream (i.e. “microwalk”) of the original gapmers fromTable 3. Gapmers were also created with various motifs, e.g. 5-10-5 MOE,3-14-3 MOE, and 2-13-5 MOE. These gapmers were tested in vitro. CulturedHepG2 cells at a density of 10,000 cells per well were transfected usinglipofectin reagent with 75 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and Factor 11 mRNA levels were measured by quantitative real-timePCR. Factor 11 mRNA levels were adjusted according to total RNA content,as measured by RIBOGREEN. Results are presented as percent inhibition ofFactor 11, relative to untreated control cells.

The in vitro inhibition data for the gapmers designed by microwalk werethen compared with the in vitro inhibition data for the gapmers fromTable 3, as indicated in Tables 4, 5, 6, 7, and 8. The oligonucleotidesare displayed according to the region on the human mRNA (GENBANKAccession No. NM_000128.3) to which they map.

The chimeric antisense oligonucleotides in Table 4 were designed as5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmersin Table 4 are the original gapmers (see Table 3) from which theremaining gapmers were designed via microwalk and are designated by anasterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein thecentral gap segment is comprised of 10 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprising5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length,wherein the central gap segment is comprised of 14 2′-deoxynucleotidesand is flanked on both sides (in the 5′ and 3′ directions) by wingscomprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides inlength, wherein the central gap segment is comprised of 132′-deoxynucleotides. The central gap is flanked on the 5′ end with awing comprising 2 nucleotides and on the 3′ end with a wing comprising 5nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), eachnucleotide in the 5′ wing segment and each nucleotide in the 3′ wingsegment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P=S) linkages. All cytidineresidues throughout each gapmer are 5-methyl cytidines. “Target startsite” indicates the 5′-most nucleotide to which the gapmer is targeted.“Target stop site” indicates the 3′-most nucleotide to which the gapmeris targeted. Each gapmer listed in Table 4 is targeted to SEQ ID NO: 1(GENBANK Accession No. NM_000128.3).

As shown in Table 4, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers,and 2-13-5 MOE gapmers targeted to the target region beginning at targetstart site 656 and ending at the target stop site 704 (i.e. nucleobases656-704) of SEQ ID NO: 1 exhibit at least 2000 inhibition of Factor 11mRNA. Many of the gapmers exhibit at least 600% inhibition. Several ofthe gapmers exhibit at least 80% inhibition, including ISIS numbers:416806, 416809, 416811, 416814, 416821, 416825, 416826, 416827, 416828,416868, 416869, 416878, 416879, 416881, 416883, 416890, 416891, 416892,416893, 416894, 416895, 416896, 416945, 416946, 416969, 416970, 416971,416972, 416973, 412203, 413467, 413468, and 413469. The following ISISnumbers exhibited at least 90 inhibition: 412203, 413467, 416825,416826, 416827, 416868, 416878, 416879, 416892, 416893, 416895, 416896,416945, 416972, and 416973. The following ISIS numbers exhibited atleast 95 inhibition: 416878, 416892, 416895, and 416896.

TABLE 4Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted tonucleobases 656 to 704 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)Target Target % SEQ ISIS No. Start Site Stop Site Sequence (5′ to 3′)inhibition Motif ID No. *412203 656 675 AATGTCTTTGTTGCAAGCGC 97 5-10-531 *413467 666 685 GGTCCACATAAATGTCTTTG 92 5-10-5 100 *413468 671 690GTCTAGGTCCACATAAATGT 83 5-10-5 101 *413469 681 700 TGCCCTTCATGTCTAGGTCC86 5-10-5 102  416868 656 675 AATGTCTTTGTTGCAAGCGC 93 3-14-3 31  416945656 675 AATGTCTTTGTTGCAAGCGC 94 2-13-5 31  416806 657 676AAATGTCTTTGTTGCAAGCG 86 5-10-5 171  416869 657 676 AAATGTCTTTGTTGCAAGCG81 3-14-3 171  416946 657 676 AAATGTCTTTGTTGCAAGCG 86 2-13-5 171  416807658 677 TAAATGTCTTTGTTGCAAGC 51 5-10-5 172  416870 658 677TAAATGTCTTTGTTGCAAGC 76 3-14-3 172  416947 658 677 TAAATGTCTTTGTTGCAAGC62 2-13-5 172  416808 659 678 ATAAATGTCTTTGTTGCAAG 55 5-10-5 173  416871659 678 ATAAATGTCTTTGTTGCAAG 28 3-14-3 173  416948 659 678ATAAATGTCTTTGTTGCAAG 62 2-13-5 173  416809 660 679 CATAAATGTCTTTGTTGCAA86 5-10-5 174  416872 660 679 CATAAATGTCTTTGTTGCAA 20 3-14-3 174  416949660 679 CATAAATGTCTTTGTTGCAA 64 2-13-5 174  416873 661 680ACATAAATGTCTTTGTTGCA 51 3-14-3 99  416950 661 680 ACATAAATGTCTTTGTTGCA71 2-13-5 99  416810 662 681 CACATAAATGTCTTTGTTGC 68 5-10-5 175  416874662 681 CACATAAATGTCTTTGTTGC 49 3-14-3 175  416951 662 681CACATAAATGTCTTTGTTGC 48 2-13-5 175  416811 663 682 CCACATAAATGTCTTTGTTG84 5-10-5 176  416875 663 682 CCACATAAATGTCTTTGTTG 75 3-14-3 176  416952663 682 CCACATAAATGTCTTTGTTG 51 2-13-5 176  416812 664 68TCCACATAAATGTCTTTGTT 59 5-10-5 177  416876 664 683 TCCACATAAATGTCTTTGTT37 3-14-3 177  416953 664 683 TCCACATAAATGTCTTTGTT 45 2-13-5 177  416813665 684 GTCCACATAAATGTCTTTGT 70 5-10-5 178  416877 665 684GTCCACATAAATGTCTTTGT 51 3-14-3 178  416954 665 684 GTCCACATAAATGTCTTTGT61 2-13-5 178  416878 666 685 GGTCCACATAAATGTCTTTG 95 3-14-3 100  416955666 685 GGTCCACATAAATGTCTTTG 75 2-13-5 100  416814 667 686AGGTCCACATAAATGTCTTT 83 5-10-5 179  416879 667 686 AGGTCCACATAAATGTCTTT92 3-14-3 179  416956 667 686 AGGTCCACATAAATGTCTTT 61 2-13-5 179  416815668 687 TAGGTCCACATAAATGTCTT 63 5-10-5 180  416880 668 687TAGGTCCACATAAATGTCTT 66 3-14-3 180  416957 668 687 TAGGTCCACATAAATGTCTT59 2-13-5 180  416816 669 688 CTAGGTCCACATAAATGTCT 79 5-10-5 181  416881669 688 CTAGGTCCACATAAATGTCT 81 3-14-3 181  416958 669 688CTAGGTCCACATAAATGTCT 43 2-13-5 181  416817 670 689 TCTAGGTCCACATAAATGTC74 5-10-5 182  416882 670 689 TCTAGGTCCACATAAATGTC 60 3-14-3 182  416959670 689 TCTAGGTCCACATAAATGTC 25 2-13-5 182  416883 671 690GTCTAGGTCCACATAAATGT 82 3-14-3 101  416960 671 690 GTCTAGGTCCACATAAATGT60 2-13-5 101  416818 672 691 TGTCTAGGTCCACATAAATG 76 5-10-5 183  416884672 691 TGTCTAGGTCCACATAAATG 69 3-14-3 183  416961 672 691TGTCTAGGTCCACATAAATG 40 2-13-5 183  416819 673 692 ATGTCTAGGTCCACATAAAT56 5-10-5 184  416885 673 692 ATGTCTAGGTCCACATAAAT 67 3-14-3 184  416962673 692 ATGTCTAGGTCCACATAAAT 77 2-13-5 184  416820 674 693CATGTCTAGGTCCACATAAA 77 5-10-5 185  416886 674 693 CATGTCTAGGTCCACATAAA74 3-14-3 185  416963 674 693 CATGTCTAGGTCCACATAAA 48 2-13-5 185  416821675 694 TCATGTCTAGGTCCACATAA 84 5-10-5 186  416964 675 694TCATGTCTAGGTCCACATAA 69 2-13-5 186  412204 676 695 TTCATGTCTAGGTCCACATA76 5-10-5 32  416888 676 695 TTCATGTCTAGGTCCACATA 76 3-14-3 32  416965676 695 TTCATGTCTAGGTCCACATA 53 2-13-5 32  416822 677 696CTTCATGTCTAGGTCCACAT 76 5-10-5 187  416889 677 696 CTTCATGTCTAGGTCCACAT60 3-14-3 187  416966 677 696 CTTCATGTCTAGGTCCACAT 64 2-13-5 187  416823678 697 CCTTCATGTCTAGGTCCACA 77 5-10-5 188  416890 678 697CCTTCATGTCTAGGTCCACA 87 3-14-3 188  416967 678 697 CCTTCATGTCTAGGTCCACA75 2-13-5 188  416824 679 698 CCCTTCATGTCTAGGTCCAC 64 5-10-5 189  416891679 698 CCCTTCATGTCTAGGTCCAC 81 3-14-3 189  416968 679 698CCCTTCATGTCTAGGTCCAC 73 2-13-5 189  416825 680 699 GCCCTTCATGTCTAGGTCCA92 5-10-5 190  416892 680 699 GCCCTTCATGTCTAGGTCCA 100 3-14-3 190 416969 680 699 GCCCTTCATGTCTAGGTCCA 80 2-13-5 190  416893 681 700TGCCCTTCATGTCTAGGTCC 90 3-14-3 102  416970 681 700 TGCCCTTCATGTCTAGGTCC88 2-13-5 102  416826 682 701 ATGCCCTTCATGTCTAGGTC 94 5-10-5 191  416894682 701 ATGCCCTTCATGTCTAGGTC 85 3-14-3 191  416971 682 701ATGCCCTTCATGTCTAGGTC 83 2-13-5 191  416827 683 702 TATGCCCTTCATGTCTAGGT93 5-10-5 192  416895 683 702 TATGCCCTTCATGTCTAGGT 95 3-14-3 192  416972683 702 TATGCCCTTCATGTCTAGGT 90 2-13-5 192  416828 684 703TTATGCCCTTCATGTCTAGG 87 5-10-5 193  416896 684 703 TTATGCCCTTCATGTCTAGG95 3-14-3 193  416973 684 703 TTATGCCCTTCATGTCTAGG 92 2-13-5 193  416829685 704 TTTATGCCCTTCATGTCTAG 72 5-10-5 194  416897 685 704TTTATGCCCTTCATGTCTAG 66 3-14-3 194  416974 685 704 TTTATGCCCTTCATGTCTAG73 2-13-5 194

The chimeric antisense oligonucleotides in Table 5 were designed as5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmerin Table 5 is the original gapmer (see Table 3) from which the remaininggapmers were designed via microwalk and is designated by an asterisk.The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gapsegment is comprised of 10 2′-deoxynucleotides and is flanked on bothsides (in the 5′ and 3′ directions) by wings comprising 5 nucleotideseach. The 3-14-3 gapmers are 20 nucleotides in length, wherein thecentral gap segment is comprised of 14 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprising3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length,wherein the central gap segment is comprised of 13 2′-deoxynucleotides.The central gap is flanked on the 5′ end with a wing comprising 2nucleotides and on the 3′ end with a wing comprising 5 nucleotides. Foreach of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOEmodification. The internucleoside linkages throughout each gapmer arephosphorothioate (P=S) linkages. All cytidine residues throughout eachgapmer are 5-methyl cytidines. “Target start site” indicates the 5′-mostnucleotide to which the gapmer is targeted. “Target stop site” indicatesthe 3′-most nucleotide to which the gapmer is targeted. Each gapmerlisted in Table 5 is targeted to SEQ ID NO: 1 (GENBANK Accession No.NM_000128.3).

As shown in Table 5, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers,and 2-13-5 MOE gapmers targeted to the target region beginning at targetstart site 738 and ending at the target stop site 762 (i.e. nucleobases738-762) of SEQ ID NO: 1 exhibit at least 450 inhibition of Factor 11mRNA. Most of the gapmers exhibit at least 6000 inhibition. Several ofthe gapmers exhibit at least 80% inhibition, including ISIS numbers:412206, 416830, 416831, 416898, 416899, 416900, 416903, 416975, 416976,416977, and 416980. The following ISIS numbers exhibited at least 90inhibition: 412206, 416831, and 416900.

TABLE 5Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted tonucleobases 738 to 762 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)Target Start Target % SEQ ID ISIS No. Site Stop Site Sequence (5′ to 3′)inhibition Motif No. *412206 738 757 CCGTGCATCTTTCTTGGCAT 93 5-10-5 34 416898 738 757 CCGTGCATCTTTCTTGGCAT 88 3-14-3 34  416975 738 757CCGTGCATCTTTCTTGGCAT 87 2-13-5 34  416830 739 758 TCCGTGCATCTTTCTTGGCA81 5-10-5 195  416899 739 758 TCCGTGCATCTTTCTTGGCA 86 3-14-3 195  416976739 758 TCCGTGCATCTTTCTTGGCA 83 2-13-5 195  416831 740 759ATCCGTGCATCTTTCTTGGC 91 5-10-5 196  416900 740 759 ATCCGTGCATCTTTCTTGGC90 3-14-3 196  416977 740 759 ATCCGTGCATCTTTCTTGGC 82 2-13-5 196  416832741 760 CATCCGTGCATCTTTCTTGG 79 5-10-5 197  416901 741 760CATCCGTGCATCTTTCTTGG 65 3-14-3 197  416978 741 760 CATCCGTGCATCTTTCTTGG76 2-13-5 197  416833 742 761 TCATCCGTGCATCTTTCTTG 65 5-10-5 198  416902742 761 TCATCCGTGCATCTTTCTTG 46 3-14-3 198  416979 742 761TCATCCGTGCATCTTTCTTG 63 2-13-5 198  416834 743 762 GTCATCCGTGCATCTTTCTT58 5-10-5 199  416903 743 762 GTCATCCGTGCATCTTTCTT 88 3-14-3 199  416980743 762 GTCATCCGTGCATCTTTCTT 87 2-13-5 199

The chimeric antisense oligonucleotides in Table 6 were designed as5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmersin Table 6 are the original gapmers (see Table 3) from which theremaining gapmers were designed via microwalk and are designated by anasterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein thecentral gap segment is comprised of 10 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprising5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length,wherein the central gap segment is comprised of 14 2′-deoxynucleotidesand is flanked on both sides (in the 5′ and 3′ directions) by wingscomprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides inlength, wherein the central gap segment is comprised of 132′-deoxynucleotides. The central gap is flanked on the 5′ end with awing comprising 2 nucleotides and on the 3′ end with a wing comprising 5nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), eachnucleotide in the 5′ wing segment and each nucleotide in the 3′ wingsegment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P=S) linkages. All cytidineresidues throughout each gapmer are 5-methylcytidines. “Target startsite” indicates the 5′-most nucleotide to which the gapmer is targeted.“Target stop site” indicates the 3′-most nucleotide to which the gapmeris targeted. Each gapmer listed in Table 6 is targeted to SEQ ID NO: 1(GENBANK Accession No. NM_000128.3).

As shown in Table 6, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers,and 2-13-5 MOE gapmers targeted to the target region beginning at targetstart site 1018 and ending at the target stop site 1042 (i.e.nucleobases 1018-1042) of SEQ ID NO: 1 exhibit at least 80% inhibitionof Factor 11 mRNA. The following ISIS numbers exhibited at least 90%inhibition: 413474, 416837, 416838, 416904, 416907, and 416908.

TABLE 6Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted tonucleobases 1018 to 1042 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)Target Target Start Stop % SEQ ID ISIS No. Site Site Sequence (5′ to 3′)inhibition Motif No. *412212 1018 1037 CCGGGATGATGAGTGCAGAT 89 5-10-5 40 416904 1018 1037 CCGGGATGATGAGTGCAGAT 90 3-14-3 40  416981 1018 1037CCGGGATGATGAGTGCAGAT 87 2-13-5 40  416835 1019 1038 ACCGGGATGATGAGTGCAGA83 5-10-5 200  416905 1019 1038 ACCGGGATGATGAGTGCAGA 85 3-14-3 200 416982 1019 1038 ACCGGGATGATGAGTGCAGA 84 2-13-5 200  416836 1020 1039AACCGGGATGATGAGTGCAG 89 5-10-5 201  416906 1020 1039AACCGGGATGATGAGTGCAG 88 3-14-3 201  416983 1020 1039AACCGGGATGATGAGTGCAG 86 2-13-5 201  416837 1021 1040CAACCGGGATGATGAGTGCA 90 5-10-5 202  416907 1021 1040CAACCGGGATGATGAGTGCA 90 3-14-3 202  416984 1021 1040CAACCGGGATGATGAGTGCA 89 2-13-5 202  416838 1022 1041GCAACCGGGATGATGAGTGC 94 5-10-5 203  416908 1022 1041GCAACCGGGATGATGAGTGC 98 3-14-3 203  416985 1022 1041GCAACCGGGATGATGAGTGC 88 2-13-5 203  413474 1023 1042AGCAACCGGGATGATGAGTG 93 5-10-5 107

The chimeric antisense oligonucleotides in Table 7 were designed as5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmerin Table 7 is the original gapmer (see Table 3) from which the remaininggapmers were designed via microwalk and is designated by an asterisk.The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gapsegment is comprised of 10 2′-deoxynucleotides and is flanked on bothsides (in the 5′ and 3′ directions) by wings comprising 5 nucleotideseach. The 3-14-3 gapmers are 20 nucleotides in length, wherein thecentral gap segment is comprised of 14 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprising3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length,wherein the central gap segment is comprised of 13 2′-deoxynucleotides.The central gap is flanked on the 5′ end with a wing comprising 2nucleotides and on the 3′ end with a wing comprising 5 nucleotides. Foreach of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOEmodification. The internucleoside linkages throughout each gapmer arephosphorothioate (P=S) linkages. All cytidine residues throughout eachgapmer are 5-methylcytidines. “Target start site” indicates the 5′-mostnucleotide to which the gapmer is targeted. “Target stop site” indicatesthe 3′-most nucleotide to which the gapmer is targeted. Each gapmerlisted in Table 7 is targeted to SEQ ID NO: 1 (GENBANK Accession No.NM_000128.3).

As shown in Table 7, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers,and 2-13-5 MOE gapmers targeted to the target region beginning at targetstart site 1062 and ending at the target stop site 1091 (i.e.nucleobases 1062-1091) of SEQ ID NO: 1 exhibit at least 20% inhibitionof Factor 11 mRNA. Many of the gapmers exhibit at least 50% inhibition,including: 412215, 413476, 413476, 416839, 416840, 416841, 416842,416843, 416844, 416845, 416846, 416847, 416909, 416910, 416911, 416912,416913, 416914, 416915, 416916, 416917, 416918, 416986, 416987, 416988,416989, 416990, 416991, 416992, 416993, 416994, 416995. The followingISIS numbers exhibited at least 80% inhibition: 412215, 413476, 413476,416839, 416840, 416841, 416842, 416843, 416844, 416845, 416910, 416911,416912, 416913, 416914, 416916, 416917, 416986, 416987, 416989, 416991,416992, 416993, and 416994. The following ISIS numbers exhibited atleast 90 inhibition: 413476, 413476, 416842, 416844, 416910, 416911,416912, 416913, 416916, 416917, and 416993.

TABLE 7Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted tonucleobases 1062 to 1091 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)ISIS Target Target % SEQ No. Start Site Stop Site Sequence (5′ to 3′)inhibition Motif ID No. *413476 1067 1086 TTGAGATTCTTTGGGCCATT 93 5-10-5109  412215 1062 1081 ATTCTTTGGGCCATTCCTGG 82 5-10-5 43  416909 10621081 ATTCTTTGGGCCATTCCTGG 78 3-14-3 43  416986 1062 1081ATTCTTTGGGCCATTCCTGG 88 2-13-5 43  416839 1063 1082 GATTCTTTGGGCCATTCCTG89 5-10-5 204  416910 1063 1082 GATTCTTTGGGCCATTCCTG 90 3-14-3 204 416987 1063 1082 GATTCTTTGGGCCATTCCTG 80 2-13-5 204  416840 1064 1083AGATTCTTTGGGCCATTCCT 85 5-10-5 205  416911 1064 1083AGATTCTTTGGGCCATTCCT 90 3-14-3 205  416988 1064 1083AGATTCTTTGGGCCATTCCT 76 2-13-5 205  416841 1065 1084GAGATTCTTTGGGCCATTCC 87 5-10-5 206  416912 1065 1084GAGATTCTTTGGGCCATTCC 92 3-14-3 206  416989 1065 1084GAGATTCTTTGGGCCATTCC 88 2-13-5 206  416842 1066 1085TGAGATTCTTTGGGCCATTC 94 5-10-5 207  416913 1066 1085TGAGATTCTTTGGGCCATTC 93 3-14-3 207  416990 1066 1085TGAGATTCTTTGGGCCATTC 76 2-13-5 207  413476 1067 1086TTGAGATTCTTTGGGCCATT 93 5-10-5 109  416914 1067 1086TTGAGATTCTTTGGGCCATT 87 3-14-3 109  416991 1067 1086TTGAGATTCTTTGGGCCATT 87 2-13-5 109  416843 1068 1087TTTGAGATTCTTTGGGCCAT 89 5-10-5 208  416915 1068 1087TTTGAGATTCTTTGGGCCAT 79 3-14-3 208  416992 1068 1087TTTGAGATTCTTTGGGCCAT 84 2-13-5 208  416844 1069 1088CTTTGAGATTCTTTGGGCCA 90 5-10-5 209  416916 1069 1088CTTTGAGATTCTTTGGGCCA 91 3-14-3 209  416993 1069 1088CTTTGAGATTCTTTGGGCCA 91 2-13-5 209  416845 1070 1089TCTTTGAGATTCTTTGGGCC 86 5-10-5 210  416917 1070 1089TCTTTGAGATTCTTTGGGCC 92 3-14-3 210  416994 1070 1089TCTTTGAGATTCTTTGGGCC 83 2-13-5 210  416846 1071 1090TTCTTTGAGATTCTTTGGGC 72 5-10-5 211  416918 1071 1090TTCTTTGAGATTCTTTGGGC 63 3-14-3 211  416995 1071 1090TTCTTTGAGATTCTTTGGGC 64 2-13-5 211  416847 1072 1091TTTCTTTGAGATTCTTTGGG 50 5-10-5 212  416919 1072 1091TTTCTTTGAGATTCTTTGGG 27 3-14-3 212  416996 1072 1091TTTCTTTGAGATTCTTTGGG 22 2-13-5 212

The chimeric antisense oligonucleotides in Table 8 were designed as5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmersin Table 8 are the original gapmers (see Table 3) from which theremaining gapmers were designed via microwalk and are designated by anasterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein thecentral gap segment is comprised of 10 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprising5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length,wherein the central gap segment is comprised of 14 2′-deoxynucleotidesand is flanked on both sides (in the 5′ and 3′ directions) by wingscomprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides inlength, wherein the central gap segment is comprised of 132′-deoxynucleotides. The central gap is flanked on the 5′ end with awing comprising 2 nucleotides and on the 3′ end with a wing comprising 5nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), eachnucleotide in the 5′ wing segment and each nucleotide in the 3′ wingsegment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P=S) linkages. All cytidineresidues throughout each gapmer are 5-methylcytidines. “Target startsite” indicates the 5′-most nucleotide to which the gapmer is targeted.“Target stop site” indicates the 3′-most nucleotide to which the gapmeris targeted. Each gapmer listed in Table 8 is targeted to SEQ ID NO: 1(GENBANK Accession No. NM_000128.3).

As shown in Table 8, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers,and 2-13-5 MOE gapmers targeted to the target region beginning at targetstart site 1275 and ending at the target stop site 1318 (i.e.nucleobases 1275-1318) of SEQ ID NO: 1 exhibit at least 70% inhibitionof Factor 11 mRNA. Many of the gapmers exhibit at least 80% inhibition,including: 412223, 412224, 412225, 413482, 416848, 416849, 416850,416851, 416852, 416853, 416854, 416855, 416856, 416857, 416858, 416859,416860, 416861, 416862, 416863, 416864, 416865, 416866, 416867, 416920,416921, 416922, 416923, 416924, 416925, 416926, 416927, 416928, 416929,416930, 416931, 416932, 416933, 416934, 416935, 416936, 416937, 416938,416939, 416940, 416941, 416942, 416943, 416944, 416997, 416998, 416999,417000, 417001, 417002, 417003, 417004, 417006, 417007, 417008, 417009,417010, 417011, 417013, 417014, 417015, 417016, 417017, 417018, 417019,and 417020. The following ISIS numbers exhibited at least 90 inhibition:412224, 416850, 416853, 416856, 416857, 416858, 416861, 416862, 416864,416922, 416923, 416924, 416925, 416926, 416928, 416931, 416932, 416933,416934, 416935, 416937, 416938, 416940, 416941, 416943, 416999, 417002,416854, and 416859.

TABLE 8Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted tonucleobases 1275 to 1318 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)ISIS Target Target % SEQ No. Start Site Stop Site Sequence (5′ to 3′)inhibition Motif ID No. *412223 1275 1294 ACAGTTTCTGGCAGGCCTCG 85 5-10-551 *412224 1285 1304 GCATTGGTGCACAGTTTCTG 93 5-10-5 52 *413482 1290 1309GGACGGCATTGGTGCACAGT 89 5-10-5 115 *412225 1295 1314GCAGCGGACGGCATTGGTGC 86 5-10-5 53  416920 1275 1294 ACAGTTTCTGGCAGGCCTCG88 3-14-3 51  416997 1275 1294 ACAGTTTCTGGCAGGCCTCG 84 2-13-5 51  4168481276 1295 CACAGTTTCTGGCAGGCCTC 86 5-10-5 213  416921 1276 1295CACAGTTTCTGGCAGGCCTC 88 3-14-3 213  416998 1276 1295CACAGTTTCTGGCAGGCCTC 88 2-13-5 213  416849 1277 1296GCACAGTTTCTGGCAGGCCT 88 5-10-5 214  416922 1277 1294GCACAGTTTCTGGCAGGCCT 94 3-14-3 214  416999 1277 1296GCACAGTTTCTGGCAGGCCT 92 2-13-5 214  416850 1278 1297TGCACAGTTTCTGGCAGGCC 93 5-10-5 215  416923 1278 1297TGCACAGTTTCTGGCAGGCC 96 3-14-3 215  417000 1278 1297TGCACAGTTTCTGGCAGGCC 89 2-13-5 215  416851 1279 1298GTGCACAGTTTCTGGCAGGC 88 5-10-5 216  416924 1279 1298GTGCACAGTTTCTGGCAGGC 96 3-14-3 216  417001 1279 1298GTGCACAGTTTCTGGCAGGC 83 2-13-5 216  416925 1280 1299GGTGCACAGTTTCTGGCAGG 98 3-14-3 114  417002 1280 1299GGTGCACAGTTTCTGGCAGG 92 2-13-5 114  416852 1281 1300TGGTGCACAGTTTCTGGCAG 84 5-10-5 217  416926 1281 1300TGGTGCACAGTTTCTGGCAG 93 3-14-3 217  417003 1281 1300TGGTGCACAGTTTCTGGCAG 89 2-13-5 217  416853 1282 1301TTGGTGCACAGTTTCTGGCA 91 5-10-5 218  416927 1282 1301TTGGTGCACAGTTTCTGGCA 87 3-14-3 218  417004 1282 1301TTGGTGCACAGTTTCTGGCA 86 2-13-5 218  416854 1283 1302ATTGGTGCACAGTTTCTGGC 90 5-10-5 219  416928 1283 1302ATTGGTGCACAGTTTCTGGC 91 3-14-3 219  417005 1283 1302ATTGGTGCACAGTTTCTGGC 79 2-13-5 219  416855 1284 1303CATTGGTGCACAGTTTCTGG 87 5-10-5 220  416929 1284 1303CATTGGTGCACAGTTTCTGG 83 3-14-3 220  417006 1284 1303CATTGGTGCACAGTTTCTGG 81 2-13-5 220  416930 1285 1304GCATTGGTGCACAGTTTCTG 87 3-14-3 52  417007 1285 1304 GCATTGGTGCACAGTTTCTG82 2-13-5 52  416856 1286 1305 GGCATTGGTGCACAGTTTCT 95 5-10-5 221 416931 1286 1305 GGCATTGGTGCACAGTTTCT 96 3-14-3 221  417008 1286 1305GGCATTGGTGCACAGTTTCT 82 2-13-5 221  416857 1287 1306CGGCATTGGTGCACAGTTTC 92 5-10-5 222  416932 1287 1306CGGCATTGGTGCACAGTTTC 92 3-14-3 222  417009 1287 1306CGGCATTGGTGCACAGTTTC 85 2-13-5 222  416858 1288 1307ACGGCATTGGTGCACAGTTT 93 5-10-5 223  416933 1288 1307ACGGCATTGGTGCACAGTTT 92 3-14-3 223  417010 1288 1307ACGGCATTGGTGCACAGTTT 81 2-13-5 223  416859 1289 1308GACGGCATTGGTGCACAGTT 90 5-10-5 224  416934 1289 1308GACGGCATTGGTGCACAGTT 90 3-14-3 224  417011 1289 1308GACGGCATTGGTGCACAGTT 86 2-13-5 224  416935 1290 1309GGACGGCATTGGTGCACAGT 92 3-14-3 115  417012 1290 1309GGACGGCATTGGTGCACAGT 72 2-13-5 115  416860 1291 1310CGGACGGCATTGGTGCACAG 88 5-10-5 225  416936 1291 1310CGGACGGCATTGGTGCACAG 89 3-14-3 225  417013 1291 1310CGGACGGCATTGGTGCACAG 86 2-13-5 225  416861 1292 1311GCGGACGGCATTGGTGCACA 92 5-10-5 226  416937 1292 1311GCGGACGGCATTGGTGCACA 93 3-14-3 226  417014 1292 1311GCGGACGGCATTGGTGCACA 87 2-13-5 226  416862 1293 1312AGCGGACGGCATTGGTGCAC 90 5-10-5 227  416938 1293 1312AGCGGACGGCATTGGTGCAC 90 3-14-3 227  417015 1293 1312AGCGGACGGCATTGGTGCAC 87 2-13-5 227  416863 1294 1313CAGCGGACGGCATTGGTGCA 83 5-10-5 228  416939 1294 1313CAGCGGACGGCATTGGTGCA 88 3-14-3 228  417016 1294 1313CAGCGGACGGCATTGGTGCA 85 2-13-5 228  416940 1295 1314GCAGCGGACGGCATTGGTGC 92 3-14-3 53  417017 1295 1314 GCAGCGGACGGCATTGGTGC82 2-13-5 53  416864 1296 1315 GGCAGCGGACGGCATTGGTG 93 5-10-5 229 416941 1296 1315 GGCAGCGGACGGCATTGGTG 95 3-14-3 229  417018 1296 1315GGCAGCGGACGGCATTGGTG 82 2-13-5 229  416865 1297 1316TGGCAGCGGACGGCATTGGT 88 5-10-5 230  416942 1297 1316TGGCAGCGGACGGCATTGGT 85 3-14-3 230  417019 1297 1316TGGCAGCGGACGGCATTGGT 84 2-13-5 230  416866 1298 1317CTGGCAGCGGACGGCATTGG 88 5-10-5 231  416943 1298 1317CTGGCAGCGGACGGCATTGG 92 3-14-3 231  417020 1298 1317CTGGCAGCGGACGGCATTGG 84 2-13-5 231  416867 1299 1318ACTGGCAGCGGACGGCATTG 83 5-10-5 232  416944 1299 1318ACTGGCAGCGGACGGCATTG 83 3-14-3 232  417021 1299 1318ACTGGCAGCGGACGGCATTG 74 2-13-5 232

Example 4: Dose-Dependent Antisense Inhibition of Human Factor 11 inHepG2 Cells

Gapmers from Example 3 (see Tables 4, 5, 6, 7, and 8), exhibiting invitro inhibition of human Factor 11, were tested at various doses inHepG2 cells. Cells were plated at a density of 10,000 cells per well andtransfected using lipofectin reagent with 9.375 nM, 18.75 nM, 37.5 nMand 75 nM concentrations of antisense oligonucleotide, as specified inTable 9. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and Factor 11 mRNA levels were measured byquantitative real-time PCR. Human Factor 11 primer probe set RTS 2966was used to measure mRNA levels. Factor 11 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. Results arepresented as percent inhibition of Factor 11, relative to untreatedcontrol cells. As illustrated in Table 9, Factor 11 mRNA levels werereduced in a dose-dependent manner in antisense oligonucleotide treatedcells.

TABLE 9 Dose-dependent antisense inhibition of human Factor 11 in HepG2cells via transfection of oligonucleotides with lipofectin 9.375 18.7537.5 75 IC₅₀ SEQ ID nM nM nM nM Motif (nM) No. 412203 33 40 62 74 5-10-524 31 412206 24 47 69 86 5-10-5 21 34 413467 35 51 62 69 5-10-5 20 100413474 29 44 57 67 5-10-5 28 107 413476 24 58 62 77 5-10-5 21 109 41682523 52 73 92 5-10-5 20 190 416826 8 36 58 84 5-10-5 29 191 416827 31 4262 77 5-10-5 23 192 416838 31 51 64 86 5-10-5 19 203 416842 18 33 62 715-10-5 31 207 416850 4 30 67 84 5-10-5 29 215 416856 21 45 58 74 5-10-527 221 416858 0 28 54 82 5-10-5 33 223 416864 18 43 62 78 5-10-5 26 229416878 22 34 60 82 5-10-5 27 100 416892 16 50 70 85 3-14-3 23 190 41689539 57 66 71 3-14-3 15 192 416896 22 39 57 81 3-14-3 27 193 416908 36 5767 76 3-14-3 16 203 416922 14 25 49 75 3-14-3 36 214 416923 36 47 60 673-14-3 23 215 416924 25 38 56 59 3-14-3 36 216 416925 13 38 59 75 3-14-330 114 416926 31 43 63 82 3-14-3 22 217 416931 44 39 57 71 3-14-3 22 221416941 33 54 63 78 3-14-3 19 229 416945 34 45 62 65 2-13-5 24 31 41696917 39 61 76 2-13-5 28 190 416972 32 40 60 69 2-13-5 26 192 416973 60 7585 87 2-13-5 3 193 416984 26 50 62 81 2-13-5 22 202 416985 17 30 47 572-13-5 49 203 416989 18 41 62 83 2-13-5 26 206 416993 15 37 50 68 2-13-536 209 416999 24 37 55 73 2-13-5 30 214 417000 35 47 58 70 2-13-5 23 215417002 35 52 67 70 2-13-5 19 114 417003 26 44 60 56 2-13-5 33 217

The gapmers were also transfected via electroporation and their dosedependent inhibition of human Factor 11 mRNA was measured. Cells wereplated at a density of 20,000 cells per well and transfected viaelectroporation with 0.7 μM, 2.2 μM, 6.7 μM, and 20 μM concentrations ofantisense oligonucleotide, as specified in Table 10. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andFactor 11 mRNA levels were measured by quantitative real-time PCR. HumanFactor 11 primer probe set RTS 2966 was used to measure mRNA levels.Factor 11 mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN. Results are presented as percent inhibition ofFactor 11, relative to untreated control cells. As illustrated in Table10, Factor 11 mRNA levels were reduced in a dose-dependent manner inantisense oligonucleotide treated cells.

TABLE 10 Dose-dependent antisense inhibition of human Factor 11 in HepG2cells via transfection of oligonucleotides with electroporation 0.7 2.26.7 20 IC₅₀ SEQ ID μM μM μM μM (μM) No. 412203 11 60 70 91 2.7 31 41220622 39 81 94 2.7 34 413467 5 31 65 89 4.2 100 413474 0 5 52 81 6.9 107413476 40 69 88 93 0.9 109 416825 27 74 92 98 1.3 190 416826 2 47 86 823.2 191 416827 37 68 87 92 1.1 192 416838 5 30 55 83 5.1 203 416842 0 1066 92 5.0 207 416850 14 25 81 91 3.4 215 416856 0 29 47 93 5.1 221416858 5 20 56 86 5.3 223 416864 32 65 78 90 1.4 229 416878 1 26 75 854.3 100 416892 14 52 82 92 2.5 190 416895 0 62 70 91 3.0 192 416896 1235 81 89 3.2 193 416908 7 58 74 89 2.8 203 416922 35 51 77 91 1.7 214416923 15 30 60 90 4.0 215 416924 22 40 63 70 4.1 216 416925 0 40 76 803.9 114 416926 47 71 91 94 0.6 217 416931 7 24 60 82 5.1 221 416941 1638 79 89 3.0 229 416945 48 70 81 88 0.6 31 416969 25 34 86 92 2.5 190416972 25 30 48 88 4.3 192 416973 20 48 86 93 2.3 193 416984 43 54 88 901.1 202 416985 12 48 45 69 5.8 203 416989 32 65 88 94 1.3 206 416993 2248 87 92 2.2 209 416999 20 42 77 88 2.8 214 417000 46 73 76 89 0.6 215417002 32 38 82 91 2.2 114 417003 0 34 75 89 3.9 217

Example 5: Selection and Confirmation of Effective Dose-DependentAntisense Inhibition of Human Factor 11 in HepG2 Cells

Gapmers exhibiting significant dose-dependent inhibition of human Factor11 in Example 4 were selected and tested at various doses in HepG2cells. Cells were plated at a density of 10,000 cells per well andtransfected using lipofectin reagent with 2.34 nM, 4.69 nM, 9.375 nM,18.75 nM, 37.5 nM, and 75 nM concentrations of antisenseoligonucleotide, as specified in Table 11. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and human Factor11 mRNA levels were measured by quantitative real-time PCR. Human Factor11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN. Results are presented as percent inhibition of human Factor11, relative to untreated control cells. As illustrated in Table 11,Factor 11 mRNA levels were reduced in a dose-dependent manner inantisense oligonucleotide treated cells compared to the control.

TABLE 11 Dose-dependent antisense inhibition of human Factor 11 in HepG2cells via transfection of oligonucleotides with lipofectin SEQ 2.34 4.699.375 18.75 37.5 75 IC50 ID nM nM nM nM nM nM Motif (nM) No. 416825 4 2239 57 79 89 5-10-5 13 190 416826 15 22 32 54 76 90 5-10-5 15 191 41683821 37 50 63 74 83 5-10-5 10 203 416850 24 31 49 55 70 77 5-10-5 13 215416858 11 35 46 61 75 77 5-10-5 11 223 416864 13 34 42 65 68 80 5-10-515 229 416892 14 34 49 70 84 93 3-14-3 9 190 416925 24 34 45 56 67 723-14-3 13 114 416999 10 26 42 62 72 80 2-13-5 14 214 417002 17 26 49 6181 84 2-13-5 12 114 417003 6 29 48 64 73 82 2-13-5 11 217

The gapmers were also transfected via electroporation and their dosedependent inhibition of human Factor 11 mRNA was measured. Cells wereplated at a density of 20,000 cells per well and transfected viaelectroporation with 625 nM, 1250 nM, 2500 nM, 5,000 nM, 10,000 nM, and20,000 nM concentrations of antisense oligonucleotide, as specified inTable 12. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and human Factor 11 mRNA levels were measured byquantitative real-time PCR. Human Factor 11 primer probe set RTS 2966was used to measure mRNA levels. Factor 11 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. Results arepresented as percent inhibition of human Factor 11, relative tountreated control cells. As illustrated in Table 12, Factor 11 mRNAlevels were reduced in a dose-dependent manner in antisenseoligonucleotide treated cells compared to the control.

TABLE 12 Dose-dependent antisense inhibition of human Factor 11 in HepG2cells via transfection of oligonucleotides with electroporation SEQ 6251250 2500 5000 10000 20000 IC50 ID nM nM nM nM nM nM (μM) No. 416825 6984 91 94 96 97 19 190 416826 67 82 89 92 95 97 33 191 416838 66 79 87 9093 96 43 203 416850 69 80 87 90 93 96 25 215 416858 65 77 87 89 93 93 44223 416864 45 74 84 87 92 94 338 229 416892 66 86 96 97 100 100 31 190416925 64 80 88 91 95 96 51 114 416999 61 82 89 94 94 97 67 214 41700259 72 86 90 94 96 156 114 417003 60 74 86 90 95 95 123 217

Example 6: Selection and Confirmation of Effective Dose-DependentAntisense Inhibition of Human Factor 11 in Cyano Primary Hepatocytes

Gapmers from Example 4 exhibiting significant dose dependent in vitroinhibition of human Factor 11 were also tested at various doses in cyanoprimary hepatocytes. Cells were plated at a density of 35,000 cells perwell and transfected via electroporation with 0.74 nM, 2.2 nM, 6.7 nM,20 nM, 60 nM, and 180 nM concentrations of antisense oligonucleotide, asspecified in Table 13. After a treatment period of approximately 16hours, RNA was isolated from the cells and human Factor 11 mRNA levelswere measured by quantitative real-time PCR. Human Factor 11 primerprobe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN. Results are presented as percent inhibition of human Factor11, relative to untreated control cells. As illustrated in Table 13,Factor 11 mRNA levels were reduced in a dose-dependent manner inantisense oligonucleotide treated cells compared to the control.

TABLE 13 Dose-dependent antisense inhibition of human Factor 11 in cyanoprimary hepatocytes SEQ 0.74 2.2 6.7 20 60 180 IC₅₀ ID nM nM nM nM nM nM(μM) No. 416825 5 22 51 61 77 84 1.0 190 416826 13 24 34 67 69 71 1.3191 416838 0 0 21 34 48 62 6.9 203 416850 2 20 24 65 69 67 1.6 215416858 2 13 22 44 63 68 3.7 223 416864 0 1 15 23 47 64 7.7 229 416892 2020 43 62 88 92 1.0 190 416925 0 9 1 48 55 76 4.4 114 416999 3 40 36 6267 82 1.3 214 417002 32 16 28 38 55 71 4.0 114 417003 12 18 19 39 58 744.1 217

Example 7: Selection and Confirmation of Effective Dose-DependentAntisense Inhibition of Human Factor 11 in HepB3 Cells by Gapmers

Gapmers exhibiting in vitro inhibition of human Factor 11 in Example 4were tested at various doses in human HepB3 cells. Cells were plated ata density of 4,000 cells per well and transfected using lipofectinreagent with 2.3 nM, 4.7 nM, 9.4 nM, 18.75 nM, 37.5 nM, and 75 nMconcentrations of antisense oligonucleotide, as specified in Table 14.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and human Factor 11 mRNA levels were measured byquantitative real-time PCR. Human Factor 11 primer probe set RTS 2966was used to measure mRNA levels. Factor 11 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. Results arepresented as percent inhibition of Factor 11, relative to untreatedcontrol cells. As illustrated in Table 14, Factor 11 mRNA levels werereduced in a dose-dependent manner in antisense oligonucleotide treatedcells compared to the control.

TABLE 14 Dose-dependent antisense inhibition of human Factor 11 in HepB3cells ISIS 2.3 4.7 9.4 18.75 37.5 75 IC₅₀ SEQ ID No. nM nM nM nM nM nM(nM) No. 416825 0 15 34 36 53 59 35 190 416826 16 28 38 55 64 66 16 191416838 23 34 43 59 71 56 11 203 416850 22 32 43 56 75 60 13 215 41685817 34 43 57 74 62 12 223 416864 24 37 42 66 76 63 9 229 416892 28 34 5068 82 72 9 190 416925 26 33 45 59 72 60 12 114 416999 19 33 42 60 71 5912 214 417002 24 30 46 57 71 65 13 114 417003 11 28 40 40 63 58 17 217

The gapmers were also transfected via electroporation and their dosedependent inhibition of human Factor 11 mRNA was measured. Cells wereplated at a density of 20,000 cells per well and transfected viaelectroporation with 41.15 nM, 123.457 nM, 370.37 nM, 1111.11 nM,3333.33 nM, and 10,000 nM concentrations of antisense oligonucleotide,as specified in Table 15. After a treatment period of approximately 16hours, RNA was isolated from the cells and human Factor 11 mRNA levelswere measured by quantitative real-time PCR. Human Factor 11 primerprobe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN. Results are presented as percent inhibition of human Factor11, relative to untreated control cells. As illustrated in Table 15,Factor 11 mRNA levels were reduced in a dose-dependent manner inantisense oligonucleotide treated cells compared to the control.

TABLE 15 Dose-dependent antisense inhibition of human Factor 11 in HepB3cells SEQ 41.15 123.457 370.37 1111.11 3333.33 10000 IC₅₀ ID nM nM nM nMnM nM (μM) No. 416825 32 40 48 75 90 92 0.16 190 416826 0 0 34 61 87 920.78 191 416838 12 9 28 40 77 88 1.20 203 416850 26 38 51 73 90 95 0.30215 416858 23 45 52 64 87 92 0.30 223 416864 4 3 6 35 75 87 2.20 229416892 9 12 28 65 89 98 0.61 190 416925 27 39 50 73 88 96 0.20 114416999 31 45 62 78 94 97 0.16 214 417002 19 0 31 47 86 93 1.20 114417003 31 0 15 43 84 92 1.50 217

Example 8: Antisense Inhibition of Murine Factor 11 in Primary MouseHepatocytes

Chimeric antisense oligonucleotides targeting murine Factor 11 weredesigned as 5-10-5 MOE gapmers targeting murine Factor 11 (GENBANKAccession No. NM_028066.1, incorporated herein as SEQ ID NO: 6). Thegapmers are 20 nucleotides in length, wherein the central gap segment iscomprised of 10 2′-deoxynucleotides and is flanked on both sides (in the5′ and 3′ directions) by wings comprising 5 nucleotides each. Eachnucleotide in each wing segment has a 2′-MOE modification. Theinternucleoside linkages throughout each gaper are phosphorothioate(P=S) linkages. All cytidine residues throughout each gapmer are5-methylcytidines. The antisense oligonucleotides were evaluated fortheir ability to reduce murine Factor 11 mRNA in primary mousehepatocytes.

Primary mouse hepatocytes were treated with 6.25 nM, 12.5 nM, 25 nM, 50nM, 100 nM, and 200 nM of antisense oligonucleotides for a period ofapproximately 24 hours. RNA was isolated from the cells and murineFactor 11 mRNA levels were measured by quantitative real-time PCR.Murine Factor 11 primer probe set RTS 2898 (forward sequenceACATGACAGGCGCGATCTCT, incorporated herein as SEQ ID NO: 7; reversesequence TCTAGGTTCACGTACACATCTTTGC, incorporated herein as SEQ ID NO: 8;probe sequence TTCCTTCAAGCAATGCCCTCAGCAATX, incorporated herein as SEQID NO: 9) was used to measure mRNA levels. Factor 11 mRNA levels wereadjusted according to total RNA content as measured by RIBOGREEN.Several of the murine antisense oligonucleotides reduced Factor 11 mRNAlevels in a dose-dependent manner.

Example 9: Cross-Reactive Antisense Inhibition of Murine Factor 11 inPrimary Mouse Hepatocytes

Antisense oligonucleotides targeted to a murine factor 11 nucleic acidwere tested for their effects on Factor 11 mRNA in vitro. Culturedprimary mouse hepatocytes at a density of 10,000 cells per well weretreated with 100 nM antisense oligonucleotide. After a treatment periodof approximately 24 hours, RNA was isolated from the cells and mouseFactor 11 mRNA levels were measured by quantitative real-time PCR.Factor 11 mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN. Results are presented as percent inhibition ofFactor 11, relative to untreated control cells.

The chimeric antisense oligonucleotides in Tables 16 were designed as5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, whereinthe central gap segment is comprised of 10 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprising5 nucleotides each. Each nucleotide in the 5′ wing segment and eachnucleotide in the 3′ wing segment has a 2′-MOE modification. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P=S) linkages. All cytidine residues throughout each gapmer are5-methylcytidines. “Mouse target start site” indicates the 5′-mostnucleotide to which the gapmer is targeted. “Mouse target stop site”indicates the 3′-most nucleotide to which the gapmer is targeted. Allthe mouse oligonucleotides listed show cross-reactivity between themouse Factor 11 mRNA (GENBANK Accession No. NM_028066.1), incorporatedherein as SEQ ID NO: 6 and the human Factor 11 mRNA (GENBANK AccessionNo. NM_000128.3), incorporated herein as SEQ ID NO: 1. “Human TargetStart Site” indicates the 5′-most nucleotide in the human mRNA (GENBANKAccession No. NM_000128.3) to which the antisense oligonucleotide istargeted. “Human Target Stop Site” indicates the 3′-most nucleotide inthe human mRNA (GENBANK Accession No. NM_000128.3) to which theantisense oligonucleotide is targeted. “Number of mismatches” indicatesthe mismatches between the mouse oligonucleotide and the human mRNAsequence.

TABLE 16Inhibition of mouse Factor 11 mRNA levels by chimeric antisense oligonucleotides having 5-10-5MOE wings and deoxy gap targeted to SEQ ID NO: 1 and SEQ ID NO: 6 MouseMouse Human Human Target Target SEQ Target Target ISIS Start Stop % IDStart Stop No. of No Site Site Sequence (5′ to 3′) Inhibition No. SiteSite mismatches 404050 379 398 TGCTTGAAGGAATATCCAGA 82 233 619 638 2404054 448 467 TAGTTCATGCCCTTCATGTC 45 234 688 707 1 404055 453 472TGTTATAGTTCATGCCCTTC 27 235 693 712 1 404066 686 705AATGTCCCTGATACAAGCCA 37 236 926 945 1 404067 691 710GGGAAAATGTCCCTGATACA 39 237 931 950 1 404083 1299 1318TGTGCAGAGTCACCTGCCAT 47 238 1533 1552 2 404087 1466 1485TTCTTGAACCCTGAAGAAAG 29 239 1709 1728 2 404089 1477 1496TGAATTATCATTTCTTGAAC 6 240 1720 1739 2 404090 1483 1502TGATCATGAATTATCATTTC 42 241 1726 1745 2

Example 10: In Vivo Antisense Inhibition of Murine Factor 11

Several antisense oligonucleotides targeted to murine Factor 11 mRNA(GENBANK Accession No. NM_028066.1, incorporated herein as SEQ ID NO: 6)showing statistically significant dose-dependent inhibition wereevaluated in vivo. BALB/c mice were treated with ISIS 404057(TCCTGGCATTCTCGAGCATT, target start site 487, incorporated herein as SEQID NO: 10) and ISIS 404071 (TGGTAATCCACTTTCAGAGG, target start site 869,incorporated herein as SEQ ID NO: 11).

Treatment

BALB/c mice were injected with 5 mg/kg, 10 mg/kg, 25 mg/kg, or 50 mg/kgof ISIS 404057 or ISIS 404071 twice a week for 3 weeks. A control groupof mice was injected with phosphate buffered saline (PBS) twice a weekfor 3 weeks. Mice were sacrificed 5 days after receiving the last dose.Whole liver was harvested for RNA analysis and plasma was collected forclotting analysis (PT and aPTT) and protein analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor11. As shown in Table 17, the antisense oligonucleotides achieveddose-dependent reduction of murine Factor 11 over the PBS control.Results are presented as percent inhibition of Factor 11, relative tocontrol.

TABLE 17 Dose-dependent antisense inhibition of murine Factor 11 mRNA inBALB/c mice mg/kg % inhibition 404057 5 40 10 64 25 85 50 95 404071 5 7210 82 25 93 50 96PT and aPTT Assay

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT)were measured using platelet poor plasma (PPP) from mice treated withISIS 404057 and ISIS 404071. PT and aPTT values provided in Table 18 arereported as International Normalized Ratio (INR) values. INR values forPT and aPTT were determined by dividing the PT or aPTT value for eachexperimental group (i.e. 5 mg/kg, 10 mg/kg, 25 mg/kg, and 50 mg/kgtreatment with ISIS 404057 or ISIS 404071) by the PT or aPTT for the PBStreated group. This ratio was then raised to the power of theInternational Sensitivity Index (ISI) of the tissue factor used. Asshown in Table 18, PT was not significantly prolonged in mice treatedwith ISIS 404057 or ISIS 404071. However, aPTT was prolonged in adose-dependent manner in mice treated with ISIS 404057 and ISIS 404071.These data suggest that antisense reduction of Factor 11 affects thecontact activation pathway, but not the extrinsic pathway of bloodcoagulation.

TABLE 18 Effect of ISIS 404071 and 404057 on PT and aPTT in BALB/c miceDose in mg/kg PT INR aPTT INR ISIS 404057 5 1.00 1.07 10 0.94 1.19 251.02 1.27 50 1.00 1.37 ISIS 404071 5 1.06 1.09 10 1.08 1.13 25 1.06 1.3550 1.02 2.08

Protein Analysis

Factor 11 proenzyme from the plasma of mice treated with ISIS 404071,was measured using a F11 assay based on clotting time. Clotting timeswere determined in duplicate with a ST4 semi-automated coagulationinstrument (Diagnostica Stago, NJ). Thirty μl of citrated sample plasmadiluted 1/20 in HEPES-NaCl buffer with BSA was incubated with 30 μl aPTTreagent (Platelet Factor 3 reagent plus particulate activator) and 30 μlof citrated plasma deficient of Factor 11 (human congential, George KingBio-Medical Inc.) at 37° C. to initiate clotting. Results wereinterpolated on a standard curve of serially diluted citrated controlmurine plasma.

As shown in Table 19, treatment with ISIS 404071 resulted in asignificant dose-dependent reduction of Factor 11 protein. Results arepresented as percent inhibition of Factor 11, relative to PBS control.

TABLE 19 Dose-dependent inhibition of murine Factor 11 protein by ISIS404071 in BALB/c mice Dose in mg/kg % Inhibition 5 39 10 67 25 89 50 96

Example 11: In Vivo Effect of Antisense Inhibition of Murine Factor 11in the FeCl₃ Induced Venous Thrombosis (VT) Model as Compared toWarfarin Treatment

ISIS 404071 and warfarin (COUMADIN) were evaluated in the FeCl₃ inducedVT mouse model. Six groups of BALB/c mice were treated with 1.25 mg/kg,2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071,administered subcutaneously twice a week for 3 weeks. Two days afterreceiving the last dose of ISIS 404071, mice were anesthetized with 150mg/kg ketamine mixed with 10 mg/kg xylazine administered byintraperitoneal injection. An additional 6 groups of BALB/c mice weretreated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kgof warfarin, administered intraperioneally daily for 6 days. Four hoursafter the last dose of warfarin, mice were anesthetized with 150 mg/kgketamine mixed with 10 mg/kg xylazine administered by intraperitonealinjection. Two control groups of BALB/c mice were treated with PBS,administered subcutaneously twice a week for 3 weeks. Two days after thelast dose of PBS, mice in both groups were anesthetized with 150 mg/kgketamine mixed with 10 mg/kg xylazine administered by intraperitonealinjection. Thrombus formation was induced with FeCl₃ in all groups ofmice except the first control group.

In mice undergoing FeCl₃ treatment, thrombus formation was induced byapplying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃solution directly on the vena cava. After 3 minutes of exposure, thefilter paper was removed. Thirty minutes after the filter paperapplication, a fixed length of the vein containing the thrombus wasdissected out for platelet analysis. Liver was collected for RNAanalysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor11. Results are presented as percent inhibition of Factor 11, relativeto PBS control. As shown in Table 20, treatment with ISIS 404071resulted in significant dose-dependent reduction of Factor 11 mRNA incomparison to the PBS control. Conversely, treatment with warfarin didnot result in significant reduction of Factor 11 as compared to the PBScontrol.

TABLE 20 Dose-dependent reduction of Factor 11 mRNA in the FeCl₃ inducedvenous thrombosis model Treatment Dose in mg/kg % inhibition Warfarin0.5 0 1 0 2 1 3 5 4 8 5 11 ISIS 404071 1.25 0 2.5 8 5 62 10 78 20 92 4096

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used toquantify platelets in the vena cava as a measure of thrombus formation.Results are presented as a percentage of PF-4 in ISIS 404071 or warfarintreated mice, as compared to the two PBS-treated control groups. Asshown in Table 21, treatment with ISIS 404071 resulted in adose-dependent reduction of PF-4 in comparison to the PBS control fordosages of 5 mg/kg and higher. Treatment with warfarin resulted in areduction of PF-4 in comparison to the PBS control for dosages of 2mg/kg and higher. Therefore, reduction of Factor 11 by the compoundsprovided herein is useful for inhibiting thrombus and clot formation.

TABLE 21 Analysis of thrombus formation by real-time PCR quantificationof PF-4 in the FeCl₃ induced venous thrombosis model Dose in mg/kg PF-4PBS − FeCl₃ 0 PBS + FeCl₃ 100 Warfarin 0.5 128 1 124 2 80 3 21 4 12 5 33ISIS 404071 1.25 143 2.5 120 5 95 10 21 20 37 40 20

Example 12: In Vivo Effect of Antisense Inhibition of Murine Factor 11Compared to Warfarin in a Tail Bleeding Assay Treatment

Tail-bleeding was measured to observe whether treatment with ISIS 404071or warfarin causes internal hemorrhage in mice. ISIS 404071 and warfarin(COUMADIN) were evaluated in the tail bleeding assay. Six groups ofBALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg,20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twicea week for 3 weeks. An additional 6 groups of BALB/c mice were treatedwith 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg ofwarfarin, administered intraperioneally daily for 6 days. A separatecontrol group of BALB/c mice was treated with PBS, administeredsubcutaneously twice a week for 3 weeks.

Tail-Bleeding Assay

Two days after the final treatment of ISIS 404071, warfarin, or PBS,mice were placed in a tail bleeding chamber. Mice were anesthetized inthe chamber with isoflurane and a small piece of tail (approximately 4mm from the tip) was cut with sterile scissors. The tail cut wasimmediately placed in a 15 mL Falcon tube filled with approximately 10mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collectedover the course of 40 minutes. The saline filled tubes were weighed bothbefore and after bleeding. The results are provided in Table 22.

Treatment with ISIS 404071 did not affect bleeding as compared to PBStreated mice. However, warfarin did increase bleeding in mice ascompared to the PBS control. Increased doses of warfarin correlatedpositively with increased blood loss. These data suggest that thehemorrhagic potential of the compounds provided herein is low,especially in comparison to warfarin. These data taken with the resultsprovided in example 11 suggest inhibition of Factor 11 with thecompounds described herein are useful for providing antithromboticactivity without associated bleeding risk.

TABLE 22 Tail bleeding assay in the FeCl₃ induced venous thrombosismodel Treatment Dose in mg/kg Blood (g) PBS 0 0.01 Warfarin 0.5 0.07 10.35 2 0.39 3 0.51 4 0.52 5 0.76 ISIS 404071 1.25 0.00 2.5 0.00 5 0.0310 0.00 20 0.06 40 0.03

Example 13: In Vivo Effect of Antisense Inhibition of Murine Factor 11Compared to Warfarin on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071or warfarin. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071,administered subcutaneously twice a week for 3 weeks. An additional 6groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneallydaily for 6 days. In a control group, BALB/c mice were treated with PBS,administered subcutaneously mice twice a week for 3 weeks.

Two days after the final dose was administered, PPP was collected and PTand aPTT assays were performed.

PT and aPTT Assay

PT and aPTT values provided in Table 16 are reported as InternationalNormalized Ratio (INR) values. INR values for PT and aPTT weredetermined by dividing the PT or aPTT value for each experimental group(i.e. 5 mg/kg, 10 mg/kg, 25 mg/kg, and 50 mg/kg treatment with ISIS404071) by the PT or aPTT for the PBS treated group. This ratio was thenraised to the power of the International Sensitivity Index (ISI) of thetissue factor used. As shown in Table 23, PT in warfarin treated mice issignificantly prolonged at every dosage. aPTT in warfarin treated micewas prolonged, particularly at dosages of 1 mg/kg and higher. ISIS404071 did not significantly affect PT, but did prolong aPTT; however,not as significantly as in warfarin treated mice. These data suggestthat ISIS 404071 affects the contact activation pathway, but not theextrinsic pathway of blood coagulation whereas warfarin affects both thecontact activation pathway and the extrinsic pathway of bloodcoagulation.

TABLE 23 Effect of ISIS 404071 and warfarin on PT and aPTT in BALB/cmice PT aPTT Treatment Dose in mg/kg INR INR Warfarin 0.5 1.41 1.10 12.03 1.31 2 2.77 1.54 3 22.76 2.90 4 6.74 2.18 5 9.20 2.29 ISIS 4040711.25 0.99 0.98 2.5 1.01 1.03 5 1.07 1.09 10 1.08 1.29 20 1.09 1.32 400.98 1.64

Example 14: In Vivo Effect of Antisense Inhibition of Murine Factor 11in the FeCl₃ Induced Venous Thrombosis (VT) Model as Compared toApixaban Treatment

ISIS 404071 and Apixaban were evaluated in the FeCl₃ induced VT mousemodel. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071,administered subcutaneously twice a week for 3 weeks. Two days afterreceiving the last dose of ISIS 404071, mice were anesthetized with 150mg/kg ketamine mixed with 10 mg/kg xylazine administered byintraperitoneal injection. An additional 6 groups of BALB/c mice weretreated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kgof Apixaban, administered subcutaneously one time. Twenty minutes afterreceiving Apixaban, mice were anesthetized with 150 mg/kg ketamine mixedwith 10 mg/kg xylazine administered by intraperitoneal injection. Twocontrol groups of BALB/c mice were treated with PBS, administeredsubcutaneously twice a week for 3 weeks. Two days after the last dose ofPBS, mice in both groups were anesthetized with 150 mg/kg ketamine mixedwith 10 mg/kg xylazine administered by intraperitoneal injection.Thrombus formation was induced with FeCl₃ in all of the mice except thefirst control group.

In mice undergoing FeCl₃ treatment, thrombus formation was induced byapplying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃solution directly on the vena cava. After 3 minutes of exposure, thefilter paper was removed. Thirty minutes after the filter paperapplication, a fixed length of the vein containing the thrombus wasdissected out for platelet analysis. Liver was collected for RNAanalysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor11. Results are presented as percent inhibition of Factor 11, relativeto PBS control. As shown in Table 24, treatment with ISIS 404071resulted in significant dose-dependent reduction of Factor 11 mRNA incomparison to the PBS control. Conversely, treatment with Apixaban didnot result in significant reduction of Factor 11 as compared to the PBScontrol.

TABLE 24 Dose-dependent reduction of Factor 11 mRNA in the FeCl₃ inducedvenous thrombosis model Dose in mg/kg % inhibition Apixaban 0.5 5 2 8 512 10 2 20 0 ISIS 404071 1.25 15 2.5 44 5 63 10 76 25 91 50 95

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used toquantify platelets in the vena cava as a measure of thrombus formation.As shown in Table 25, treatment with ISIS 404071 resulted in reductionof PF-4 in comparison to the PBS control. Treatment with Apixaban alsoresulted in reduction of PF-4, in comparison to the PBS control. Resultsare presented as a percentage of PF-4 in ISIS 404071 or Apixaban treatedmice, as compared to the two PBS-treated control groups.

TABLE 25 Analysis of thrombus formation by real-time PCR quantificationof PF-4 in the FeCl₃ induced venous thrombosis model Treatment Dose inmg/kg PF-4 PBS − FeCl₃ 0 PBS + FeCl₃ 100 Apixaban 0.5 67 2 46 5 15 10 520 26 ISIS 404071 1.25 42 2.5 87 5 60 10 28 25 14 50 4

Example 15: In Vivo Effect of Antisense Inhibition of Murine Factor 11Compared to Apixaban in the Tail Bleeding Assay Treatment

Tail bleeding was measured to observe whether treatment with ISIS 404071or warfarin causes internal hemorrhage in mice. ISIS 404071 and Apixabanwere evaluated in the tail bleeding model. Six groups of BALB/c micewere treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3weeks. An additional 6 groups of BALB/c mice were treated with 0.5mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of Apixaban,administered in a single subcutaneous dose. A separate control group ofBALB/c mice was treated with PBS, administered subcutaneously twice aweek for 3 weeks.

Tail-Bleeding Assay

Two days after the final treatment of ISIS 404071, Apixaban, or PBS,mice were placed in a tail bleeding chamber. Mice were anesthetized inthe chamber and a small piece of tail (approximately 4 mm from the tip)was cut with sterile scissors. The cut tail was immediately placed in a15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffersolution warmed to 37° C. The blood was collected over the course of 40minutes. The saline filled tubes were weighed before and after bleeding.

As shown in Table 26, treatment with ISIS 404071 did not affect bleedingas compared to PBS treated mice. However, Apixaban did increase bleedingin mice as compared to the PBS control. Increased doses of Apixabancorrelated positively with increased blood loss. These data suggest thatthe hemorrhagic potential of the compounds provided herein is low,especially in comparison to Apixaban. These data taken with the resultsprovided in example 14 suggest inhibition of Factor 11 with thecompounds described herein are useful for providing antithromboticwithout associated bleeding risk.

TABLE 26 Tail bleeding assay in BABL/c mice mg/kg Blood (g) PBS 0 0.06Apixaban 0.5 0.03 2 0.34 5 0.37 10 0.40 20 0.52 ISIS 404071 1.25 0.002.5 0.03 5 0.00 10 0.04 25 0.01 50 0.01

Example 16: Ex Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with LOVENOX Treatment

Three groups of BALB/c mice were treated with 10 mg/kg, 20 mg/kg, or 40mg/kg of ISIS 404071, administered subcutaneously twice a week for 3weeks. A control mouse group was treated with PBS, administered twice aweek for 3 weeks. Five days after the last dose, the mice weresacrificed and plasma was collected. The low-molecular-weight (LMW)heparin, LOVENOX, was administered to the plasma ex vivo at varyingconcentrations of 0 μg/ml, 2.5 μg/ml, 5.0 μg/ml, and 7.5 μg/ml. PT andaPTT were measured 20 minutes after LOVENOX was administered.

PT and aPTT Assay

As shown in Table 27, treatment with LOVENOX increases PT in adose-dependent manner. Treatment with ISIS 404071 does not significantlyincrease PT. PT is not significantly affected by treatment with ISIS404071. There is no evidence of a combinational effect on PT in ISIS404071 and LOVENOX treated plasma.

TABLE 27 Effect of combination of ISIS 404071 and LOVENOX on PT INR inmurine plasma ISIS 404071 LOVENOX (mg/ml) (mg/kg) 0 2.5 5.0 7.5 0 1.001.02 1.10 1.12 10 0.97 1.07 1.10 1.12 20 1.00 1.10 1.07 1.10 40 0.971.02 1.07 1.10

As shown in Table 28, treatment with LOVENOX increases aPTT in adose-dependent manner. Treatment with ISIS 404071 also increases aPTT ina dose-dependent manner. Furthermore, the combined treatment of ISIS404071 and LOVENOX appears to have a synergistic effect on aPTT.

TABLE 28 Effect of combination of ISIS 404071 and LOVENOX on aPTT INR inmurine plasma ISIS 404071 LOVENOX (mg/ml) mg/kg 0 2.5 5.0 7.5 0 1.001.53 2.10 2.70 10 1.14 1.76 2.39 3.20 20 1.28 1.95 2.83 3.65 40 1.522.66 n.d. 4.78 n.d. = no data

Example 17: In Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with LOVENOX in the FeCl₃ Induced Venous Thrombosis (VT)Model Treatment

The combination of ISIS 404071 and LOVENOX were evaluated in the FeCl₃induced VT mouse model. Four groups of BALB/c mice were treated with 15mg/kg, 30 mg/kg, 45 mg/kg, or 60 mg/kg of LOVENOX, administeredsubcutaneously once daily for 3 days. An additional 4 groups of BALB/cmice were treated with 20 mg/kg of ISIS 404071, administeredsubcutaneously twice weekly for 3 weeks. After the last dose of ISIS404071, mice were treated with 15 mg/kg, 30 mg/kg, 45 mg/kg, or 60 mg/kgof LOVENOX, administered subcutaneously once daily for 3 days. Twocontrol groups of BALB/c mice were treated with PBS, administeredsubcutaneously twice a week for 3 weeks. Thrombus formation was inducedwith FeCl₃ in all of the mice except the first control group. All micewere anesthetized with 150 mg/kg of ketamine mixed with 10 mg/kg ofxylazine administered by intraperitoneal injection.

In mice undergoing FeCl₃ treatment, thrombus formation was induced byapplying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃solution directly on the vena cava. After 3 minutes of exposure, thefilter paper was removed. Thirty minutes after the filter paperapplication, a fixed length of the vein containing the thrombus wasdissected out for platelet analysis.

Quantification of Platelet Composition

Real-time PCR quantification of PF-4 was used to quantify platelets inthe vena cava as a measure of thrombus formation. As shown in Table 29,treatment with LOVENOX resulted in a reduction of PF-4 in comparison tothe PBS control. Treatment with LOVENOX in combination with ISIS 404071resulted in a higher reduction of PF-4 in comparison to LOVENOX alone.

TABLE 29 Analysis of thrombus formation by real-time PCR quantificationof PF-4 in the FeCl₃ induced venous thrombosis model Treatment mg/kgPF-4 PBS − FeCl₃ 0 PBS + FeCl₃ 100 LOVENOX 15 57 30 33 45 10 60 5LOVENOX (+ISIS 15 0 404071) 30 0 45 11 60 5

Example 18: In Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with LOVENOX on Bleeding Treatment

Tail-bleeding was measured to observe whether treatment with ISIS 404071and LOVENOX causes internal hemorrhage in mice. ISIS 404071 wasadministered subcutaneously at a dosage of 20 mg/kg twice a week for 3weeks to 4 groups of BALB/c mice, and LOVENOX was administeredsubcutaneously at varying dosages of 15 mg/kg, 30 mg/kg, 45 mg/kg, and60 mg/kg once daily on the last three days of ISIS 404071 treatment. Ina fifth group, ISIS 404071 was administered subcutaneously to BALB/cmice at a dosage of 20 mg/kg twice a week for 3 weeks. In a sixth group,PBS was administered subcutaneously twice a week for three weeks toBALB/c mice, as a control.

Tail-Bleeding Assay

Two days after receiving their final treatment, mice were placed in atail bleeding chamber. Mice were anesthetized in the chamber withisoflurane and a small piece of tail (approximately 4 mm from the tip)was cut with sterile scissors. The cut tail was immediately placed in a15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffersolution warmed to 37° C. The blood was collected over the course of 40minutes. The saline filled tubes were weighed both before and afterbleeding.

As shown in Table 30, LOVENOX increased bleeding in mice compared to thePBS treated mice. Increased doses of LOVENOX correlated positively withincreased blood loss. ISIS 404071 combined with LOVENOX did notsignificant increase bleeding beyond the increased blood loss shown inLOVENOX only treated mice.

TABLE 30 Tail bleeding assay comparing LOVENOX and the combination ofLOVENOX and ISIS 404071 Dose in Blood mg/kg (g) PBS 0.05 LOVENOX 15 0.1130 0.20 45 0.27 60 0.47 LOVENOX (+ISIS 15 0.14 404071) 30 0.19 45 0.3660 0.61

Example 19: In Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with LOVENOX on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071in combination with LOVENOX. In the first cohort, ISIS 404071 wasadministered subcutaneously to BALB/c mice at a dosage of 25 mg/kg twicea week for 3 weeks. Plasma was collected from these mice 5 days afterreceiving the last dose of ISIS 404071. In the second cohort, LOVENOXwas administered subcutaneously to BALB/c mice at a dosage of 20 mg/kgonce daily for three days. Plasma was collected from these mice 4 hoursafter receiving the last dose of LOVENOX. In the third cohort, ISIS404071 was administered subcutaneously to BALB/c mice at a dosage of 20mg/kg twice a week for 3 weeks, and 2 days after receiving the last doseof ISIS 404071, LOVENOX was administered subcutaneously at a dosage of20 mg/kg once daily. Plasma was collected from these mice 4 hours afterthe last dose of LOVENOX. In a fourth cohort, PBS was administeredsubcutaneously twice a week for three weeks, as a control. Plasma wascollected from these mice 5 days after the last dose.

PT and aPTT Assay

PT and aPTT values provided in Table 31 are reported as InternationalNormalized Ratio (INR) values. As shown in Table 31, PT is notsignificantly affected by treatment with ISIS 404071, LOVENOX, ortreatment with ISIS 40471 combined with LOVENOX. These data suggest thatthere is no combinational effect on PT by ISIS 404071 combined withLOVENOX. Also shown in Table 31, treatment with LOVENOX and treatmentwith ISIS 404071 combined with LOVENOX increase aPTT. These data suggestthat the combined treatment of ISIS 404071 and LOVENOX has an additiveeffect on aPTT.

TABLE 31 Effect of combination of ISIS 404071 and LOVENOX on PT and aPTTin murine plasma PT INR aPTT INR ISIS 404071 0.95 1.31 LOVENOX 1.04 2.04404071 + LOVENOX 1.04 2.58

Example 20: In Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with Apixaban on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071in combination with Apixaban. In the first cohort, ISIS 404071 wasadministered subcutaneously to BALB/c mice at a dosage of 25 mg/kg twicea week for 3 weeks. Plasma was collected from these mice 5 days afterreceiving the last dose of ISIS 404071. In the second cohort, Apixabanwas administered subcutaneously to BALB/c mice at a dosage of 6 mg/kgtwice daily for three days. Plasma was collected from these mice 20minutes after receiving the last dose of Apixaban. In the third cohort,ISIS 404071 was administered subcutaneously to BALB/c mice at a dosageof 20 mg/kg twice a week for 3 weeks, and Apixaban was administeredsubcutaneously at a dosage of 6 mg/kg twice daily on the last three daysof ISIS 404071 treatment. Plasma was collected from these mice 20minutes after receiving the last dose of Apixaban. In a fourth cohort,PBS was administered subcutaneously twice a week for three weeks, as acontrol. Plasma was collected 5 days after the last dose of PBS.

PT and aPTT Assay

PT and aPTT values provided in Table 32 are reported as InternationalNormalized Ratio (INR) values. As shown in Table 32, PT is notsignificantly affected by treatment with ISIS 404071. However, Apixabanand Apixaban combined with ISIS 404071 increased PT. Also shown in Table32, Apixaban, ISIS 404071, and ISIS 404071 combined with Apixabanincrease aPTT.

TABLE 32 Effect of combination of ISIS 404071 and Apixaban on PT andaPTT in murine plasma PT INR aPTT INR ISIS 404071 0.95 1.31 Apixaban3.25 1.44 404071 + Apixaban 3.50 2.26

Example 21: In Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with Warfarin on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071in combination with warfarin. Two groups of BALB/c mice were treatedwith either 25 mg/kg or 50 mg/kg of ISIS 404071, administeredsubcutaneously twice a week for 3 weeks. Plasma was collected from eachgroup 5 days after the last dose was administered. In a third group,BALB/c mice were treated with 2 mg/kg of warfarin once daily for 5 days.Plasma was collected 6 hours after the last dose of warfarin wasadministered. Two additional groups of BALB/c mice were treated witheither 25 mg/kg or 50 mg/kg of ISIS 404071, administered subcutaneouslytwice a week for 3 weeks and warfarin was administered subcutaneously ata dosage of 2 mg/kg once daily on the last 5 days of ISIS 404071treatment. Plasma was collected from each group 6 hours after the lastwarfarin treatment. In a final group of BALB/c mice, PBS wasadministered subcutaneously twice a week for three weeks, as a control.Plasma was collected 5 days after the last PBS treatment.

PT and aPTT Assay

PT and aPTT values provided in Table 33 are reported as InternationalNormalized Ratio (INR) values. As shown in Table 33, PT is not affectedby treatment with PBS or ISIS 404071 at either dosage. However,treatment with 2 mg/kg warfarin, 25 mg/kg ISIS 404071 in combinationwith 2 mg/kg warfarin, and 50 mg/kg ISIS 404071 in combination with 2mg/kg warfarin increase PT. These data suggest that the combinedtreatment of ISIS 404071 and warfarin has an additive effect on PT. Alsoshown in Table 33, aPTT is affected by treatment with ISIS 404071 andwarfarin. The combination of ISIS 404071 and warfarin show an increasein aPTT greater than either drug alone. These data suggest that thecombined treatment of ISIS 404071 and warfarin has a synergistic effecton aPTT.

TABLE 33 Effect of combination of ISIS 404071 and warfarin on PT andaPTT in murine plasma Dose in aPTT mg/kg PT INR INR ISIS 404071 25 0.981.37 50 0.93 1.49 Warfarin 2 21.33 2.52 ISIS 404071 (+Warfarin) 25 25.774.45 50 36.33 4.75

Example 22: In Vivo Antithrombotic Effect of Antisense Inhibition ofMurine Factor 11 on Mesenteric Vein Thrombosis in Mice Treatment

In a first cohort, ISIS 404071 was administered subcutaneously toC57BL/6 mice twice a week for three weeks at a dose of 50 mg/kg. In asecond cohort, a control oligonucleotide, ISIS 405277(AAGGACCTACACTATGGAAT; antisense oligonucleotide for Factor 2),incorporated herein as SEQ ID NO: 12 was administered subcutaneously toC57Bl/6 mice twice a week for three weeks at a dose of 50 mg/kg.

Platelet Preparation

Blood was collected from the retro-orbital venous plexus of naïveC57BL/6 mice by puncture and collected in polypropylene tubes containing300 μl of heparin (30 U/ml). Platelet rich plasma (PRP) was obtained bycentrifugation at 1000 rpm for 5 min. The PRP was transferred to freshtubes containing 2 μl of Prostaglandin I₂ (PGI₂) (2 μg/ml) and incubatedat 37° C. for 5 min. After centrifugation at 2600 rpm, pellets wereresuspended in 1 ml modified Tyrode's-HEPES buffer (137 mM NaCl, 0.3 mMNa₂HPO₄, 2 mM KCl, 12 mM NaHCO₃, 5 mM HEPES, 5 mM glucose, 0.35% BSA, pH7.2) containing 2 μl of PGI₂ and incubated at 37° C. for 5 min. Thesuspended pellet was centrifuged at 2600 rpm for 5 min. To remove PGI₂,the washing step was repeated twice and platelets were fluorescentlylabeled with calcein AM 2.5 μg/mL (Molecular Probes, Eugene, Oreg.) for10 min at room temperature.

Intravital Microscopy for Thrombosis

Fluorescently-labeled platelets were injected intravenously in ISIS404071 treated and control oligonucleotide treated C57BL/6 mice. Themice were anaesthetized with 2.5% avertin, and an incision was madethrough the abdominal wall to expose mesenteric veins 250-300-μm indiameter and having a shear rate of approximately 150 s⁻¹. The exposedmesentery was kept moist throughout the experiment by periodicsuperfusion with warmed (37° C.) PBS. The mesentery was transluminatedwith a 12V, 100 W, DC stabilized source. Veins were visualized using aZeiss (Germany) Axiovert 135 inverted microscope (Objective 32X)connected to an SVHS video recorder (AG-6730; Panasonic, Tokyo, Japan)using a CCD video camera (Hamamatsu Photonic Systems, Hamamatsu City,Japan). Centerline erythrocyte velocity (V_(rbc)) was measured using anoptical Doppler velocimeter (Microcirculation Research Institute, TexasA&M College of Medicine, College Station, Tex.). Venular shear rate (τ)was calculated based on Poiseuille's Law for a newtonian fluid,τ=8(V_(mean)/D_(v)), where D_(v) is the diameter of the venule andV_(mean) is estimated from the measured V_(rbc) using the empiricalcorrelation V_(mean)=V_(rbc)/1.6.

Results Analysis

Mesenteric vein thrombosis was performed two days after the lastantisense oligonucleotide injection. Thrombosis was induced by applyingWhatman paper soaked in a 10% FeCl₃ solution for 5 minutes on themesenteric vein. The vein was monitored for 40 minutes, or untilocclusion. The elapsed time before the first thrombus 30-50 μm indiameter and the elapsed time before blood stopped flowing for 30seconds were observed.

Thrombus formation (30 μm in diameter) occurred in mice treated withISIS 404071 at 14.8±1.7 minutes. Thrombus formation (30 μm in diameter)occurred in control mice at 8.9±0.6 minutes. Occlusive thrombi formed incontrol mice at 19.3±0.8 min and all injured venules occluded. Incontrast, the majority of the veins in ISIS 404071 treated mice did notocclude when observation was terminated 40 minutes after injury andthose veins showing occlusion. The only vein showing occlusion in theISIS 404071 treated mice occluded at 29.5 minutes and reopened after 5minutes, prior to the end of the study.

Example 23: In Vivo Sense-Oligonucleotide-Antidote for AntisenseInhibition of Murine Factor 11 in BALB/c Mice Treatment

The effect of the specific sense oligonucleotide to ISIS 404071 as anantidote was tested in BALB/c mice. In a first cohort, ISIS 404071 wasadministered subcutaneously to BALB/c mice twice a week for three weeksat a dose of 40 mg/kg. In a second cohort, ISIS 404057 was administeredsubcutaneously to BALB/c mice twice a week for three weeks at a dose of40 mg/kg. The ISIS 404071 specific antidote, ISIS 418026(CCTCTGAAAGTGGATTACCA; complementary to ISIS 404071), incorporatedherein as SEQ ID NO: 13, was administered to both cohorts subcutaneouslyin a single injection of 90 mg/kg 48 hours after the final treatment ofISIS 404071 or 404057. In a third cohort, ISIS 404071 was administeredsubcutaneously to BALB/c mice twice a week for three weeks at a dose of40 mg/kg. Following the last treatment of ISIS 404071, mice wereinjected subcutaneously injected with PBS. In a fourth cohort, ISIS404057 was administered subcutaneously to BALB/c mice twice a week forthree weeks at a dose of 40 mg/kg. Following the last treatment of ISIS404057, mice were injected subcutaneously injected with PBS. Followingantidote administration, a set of 4 mice from each cohort weresacrificed at 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days.Whole liver was collected for RNA analysis and PPP was collected foraPTT analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor11. Results are presented as percent inhibition of Factor 11, relativeto PBS control. As shown in Table 34, mice treated with ISIS 404071without antidote showed progressive decrease in inhibition over the 14day observation period. However, mice treated with ISIS 404071 andantidote showed an accelerated decrease in inhibition over the 14 dayobservation period in comparison to mice which did not receive antidote.Also shown in Table 34, treatment with ISIS 418026 had no effect oninhibition of Factor 11 mRNA expression in ISIS 404057 treated mice.

TABLE 34 Percent inhibition of mouse Factor 11 mRNA compared to PBScontrol 12 1 2 3 7 14 hours day days days days days ISIS 404071 93 90 8988 81 67 ISIS 404071 + 90 87 72 66 57 31 ISIS 418026 ISIS 404057 n.d.n.d. n.d. 95 n.d. n.d. ISIS 404057 + ISIS n.d. n.d. n.d. 97 n.d. n.d.418026 n.d. = no dataaPTT Assay

As shown in Table 35, mice treated with ISIS 404071 and antidote (ISIS418026) showed progressive decrease of aPTT over the 14 day observationperiod compared to mice treated with ISIS 404071 without antidote.

TABLE 35 Effect of antidote treatment on aPTT INR 12 hours 1 day 2 day 3day 7 day 14 day ISIS 404071 1.51 1.30 1.35 1.27 1.18 1.05 ISIS 404071 +1.45 1.23 1.16 1.15 1.10 0.95 ISIS 418026

Example 24: In Vivo Factor 7a Protein-Antidote for Antisense Inhibitionof Murine Factor 11 in BALB/c Mice Treatment

The effect of human Factor 7a (Factor VIIa) protein as an antidote forISIS 404071 was tested in BALB/c mice. Two experimental groups of BALB/cmice were treated with 20 mg/kg of ISIS 404071, administeredsubcutaneously twice a week for 3 weeks. Two control groups of BALB/cmice were treated with PBS, administered subcutaneously twice a week for3 weeks. Thrombus formation was induced with FeCl₃ in all of the miceexcept the first control group. Fifteen minutes before FeCl₃ treatment,the first experimental group was treated with 5 μg/kg of human Factor 7aprotein antidote (product no. 407act, American Diagnostica Inc.). Twodays after their last dose, all mice were anesthetized with 150 mg/kg ofketamine mixed with 10 mg/kg of xylazine administered by intraperitonealinjection.

In mice undergoing FeCl₃ treatment, thrombus formation was induced byapplying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃solution directly on the vena cava. After 3 minutes of exposure, thefilter paper was removed. Thirty minutes after the filter paperapplication, a fixed length of the vein containing the thrombus wasdissected out for platelet analysis.

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used toquantify platelets in the vena cava as a measure of thrombus formation.Results are presented as a percentage of PF-4 in antidote treated anduntreated mice, as compared to the two PBS-treated control groups. Asshown in Table 36, animals treated with human Factor 7a protein antidoteexpressed more PF-4 in comparison to animals treated with ISIS 404071alone. These data indicate that human Factor 7a is successful inrescuing the effect of antisense oligonucleotide inhibition.

TABLE 36 Analysis of thrombus formation by real-time PCR quantificationof PF-4 in the FeCl₃ induced venous thrombosis model Treatment PF-4 PBS− FeCl₃ 0 PBS + FeCl₃ 100 ISIS 404071 18 ISIS 404071 + hFV7a 68

Example 25: In Vivo Antisense Inhibition of Murine Factor 11 in theCollagenase-Induced Intracerebral Hemorrhage Model Treatment

ISIS 404071 and warfarin (COUMADIN) were examined in thecollegenase-induced intracerebral hemorrhage model. In a first cohort,ISIS 404071 was administered subcutaneously to BALB/c mice twice a weekfor two weeks at a dose 40 mg/kg. In a second cohort, warfarin wasadministered intraperioneally to mice twice a week for two weeks at adose of 2 mg/kg. In a third cohort, ISIS 421208 (TCGGAAGCGACTCTTATATG, 8mismatches to murine Factor 11, incorporated herein as SEQ ID NO: 14)was administered subcutaneously to BALB/c mice twice a week for twoweeks at a dose 40 mg/kg. In a fourth cohort, PBS was administered toBALB/c mice twice a week for two weeks.

Two days after receiving their final dose, all mice in all cohorts wereanesthetized with 5 μg/g of avertin. Next, the mice were injected at −1mm AP, 1 mm R ML, −4 mm DV from bregma flat skull with a 10 μL Hamiltonsyringe containing 0.075 U collagenase (150 U/mL). Collagenase wasdelivered over 5 minutes and the needle was kept in place for anadditional 5 minutes to prevent reflux. The mice were then analyzed forhemorrhagic size, neurologic deficit score, and mortality.

Table 37 presents the hemorrhage volume detected in mice aftercollagenase treatment, Table 38 presents the neurologic deficit score ofthe mice, and Table 39 presents the mortality rate of the mice.Neurological deficit is measured by a standard scoring system where nodeficiency is zero and severe deficit is five. Collectively, the datasuggest that ISIS 404071 did not have a significant effect on thehemorrhagic size, neurologic deficit score, or mortality of the mice.Thus, risk of intracerebral hemorrhage (a risk factor for warfarintreated individuals) is significantly reduced in ISIS 404071 treatedmice in comparison to warfarin treated mice.

TABLE 37 Hemorrhagic volume after collagenase treatment Volume (mm³) PBS51 ISIS 421208 41 ISIS 404071 38

TABLE 38 Neurologic Deficit Score after collagenase treatment Score PBS2.4 ISIS 421208 2.0 ISIS 404071 3.8

TABLE 39 Mortality after collagenase treatment % mortality PBS 0 ISIS421208 0 ISIS 404071 20 Warfarin 80

Example 26: In Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with PLAVIX in the FeCl₃ Induced Venous Thrombosis (VT)Model Treatment

The combination of ISIS 404071 and PLAVIX was evaluated in the FeCl₃induced VT mouse model. Four groups of eight BALB/c mice, weighingapproximately 25 g each, were treated with 6.25 mg/kg, 12.50 mg/kg,25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Mice were given two doses ofPLAVIX on day one and one dose of PLAVIX on day two, two hours beforesurgery.

An additional four groups of eight BALB/c mice, weighing approximately25 g each, were treated with 20 mg/kg of ISIS 404071, administeredsubcutaneously twice a week for three weeks. After the last dose of ISIS404071, mice were treated with 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or50.00 mg/kg of PLAVIX. Two doses of PLAVIX were administered to the miceon day one and one dose of PLAVIX was administered on day two, two hoursbefore surgery.

Two control groups of eight BALB/c mice, weighing approximately 25 geach, were not treated with ISIS 404071 or PLAVIX. An additional twocontrol groups of eight BALB/c mice, weighing approximately 25 g each,were treated with 20 mg/kg of ISIS 404071, administered subcutaneouslytwice a week for three weeks, but were not treated with PLAVIX. Thrombusformation was induced with FeCl₃ in all of the mice except the first andthird control groups. All mice were anesthetized with 150 mg/kg ofketamine mixed with 10 mg/kg of xylazine administered by intraperitonealinjection.

In mice undergoing FeCl₃ treatment, thrombus formation was induced byapplying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃solution directly on the inferior vena cava. After 3 minutes ofexposure, the filter paper was removed. Thirty minutes after the filterpaper application, a fixed length of the vein containing the thrombuswas dissected out for platelet analysis.

Quantification of Platelet Composition

Real-time PCR quantification of PF-4 was used to quantify platelets inthe vena cava as a measure of thrombus formation. As shown in Table 40,treatment with PLAVIX resulted in a reduction of PF-4 in comparison tothe PBS control. Treatment with PLAVIX in combination with ISIS 404071resulted in a higher reduction of PF-4 in comparison to PLAVIX alone.Therefore, the combination of anti-platelet therapy with Factor 11 ASOincreases antithrombotic activity. Data is presented as percent of PF-4mRNA as compared to the PBS+ FeCl₃ control.

TABLE 40 Analysis of thrombus formation by real-time PCR quantificationof PF-4 in the FeCl3 induced venous thrombosis model ISIS 404071 PLAVIXTreatment mg/kg mg/kg PF-4 PBS − FeCl₃ 0 0 29 PBS + FeCl₃ 0 0 100 PLAVIXonly 0 6.25 59 0 12.50 37 0 25.00 30 0 50.00 30 ISIS 404071 − FeCl₃ 20 027 ISIS 404071 + FeCl₃ 20 0 40 PLAVIX (+ISIS 20 6.25 35 404071) 20 12.5038 20 25.00 25 20 50.00 35

Example 27: In Vivo Effect of Antisense Inhibition of Murine Factor 11in Combination with PLAVIX on Bleeding Treatment

Tail-bleeding was measured to observe whether treatment with ISIS 404071in combination with PLAVIX causes an increase in bleeding tendency. ISIS404071 was administered subcutaneously at a dosage of 20 mg/kg twice aweek for 3 weeks to 5 groups of eight BALB/c mice. After the last doseof ISIS 404071, mice were treated with 0 mg/kg, 6.25 mg/kg, 12.50 mg/kg,25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Two doses of PLAVIX wereadministered to the mice on day one and one dose of PLAVIX wasadministered on day two, two hours before bleeding.

An additional 5 groups of eight BABL/c mice were treated similarly,except they did not receive ISIS 404071 injections.

Tail-Bleeding Assay

Two hours after receiving their final treatment, mice were placed in atail bleeding chamber. Mice were anesthetized in the chamber withisoflurane and a small piece of tail (approximately 4 mm from the tip)was cut with sterile scissors. The cut tail was immediately placed in a15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffersolution warmed to 37° C. The blood was collected for the course of 40minutes. The saline filled tubes were weighed both before and afterbleeding.

Taken with the results of Example 26, these data show that thecombination of anti-platelet therapy with Factor 11 ASO increasesantithrombotic activity without increased bleeding risk.

TABLE 41 Tail bleeding assay comparing PLAVIX and the combination ofPLAVIX and ISIS 404071 ISIS 404071 PLAVIX Treatment mg/kg mg/kg Blood(g) No treatment 0 0 0.040 PLAVIX only 0  6.25 mg/kg 0.075 0 12.50 mg/kg0.205 0 25.00 mg/kg 0.524 0 50.00 mg/kg 0.628 ISIS 404071 only 20 mg/kg0 0 PLAVIX (+ISIS 20 mg/kg  6.25 mg/kg 0.065 404071) 20 mg/kg 12.50mg/kg 0.300 20 mg/kg 25.00 mg/kg 0.401 20 mg/kg 50.00 mg/kg 0.577

Example 28: In Vivo Effect of a Factor Xa Small Molecule Inhibitor inCombination with PLAVIX on Bleeding Treatment

Tail-bleeding was measured to observe whether treatment with a Factor10a small molecule in combination with PLAVIX causes an increase inbleeding tendency. Five groups of eight BALB/c mice were treated with 0mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX.Mice were given two doses of PLAVIX on day one and one dose of PLAVIX onday two, two hours before bleeding.

An additional five groups of eight BALB/c mice were treated with 0mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX.Mice were given two doses of PLAVIX on day one and one dose of PLAVIX onday two, two hours before bleeding. These mice were also treated with0.5 mg/kg of Apixaban, a small molecule Factor 10a inhibitor,intraperitoneally one time 20 minutes before bleeding.

Tail-Bleeding Assay

Two hours after receiving their final treatment, mice were placed in atail bleeding chamber. Mice were anesthetized in the chamber withisoflurane and a small piece of tail (approximately 4 mm from the tip)was cut with sterile scissors. The cut tail was immediately placed in a15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffersolution warmed to 37° C. The blood was collected for the course of 40minutes. The saline filled tubes were weighed both before and afterbleeding.

As shown below in Table 42, these data show that the combination ofanti-platelet therapy with a small molecule Factor 10a inhibitor, suchas Apixaban, increases bleeding risk. Therefore, treatment with thecombination of anti-platelet therapy with a Factor 11 ASO provides abetter safety profile in comparison to the safety profile of acombination of anti-platelet therapy with a small molecule Factor 10ainhibitor.

TABLE 42 Tail bleeding assay comparing PLAVIX, Apixaban, and thecombination of PLAVIX and Apixaban Apixaban PLAVIX Treatment mg/kg mg/kgBlood (g) No treatment 0 0 0.002 PLAVIX only 0  6.25 mg/kg 0.061 0 12.50mg/kg 0.149 0 25.00 mg/kg 0.246 0 50.00 mg/kg 0.258 Apixaban only 0.5mg/kg 0 0.004 PLAVIX (+Apixaban) 0.5 mg/kg  6.25 mg/kg 0.258 0.5 mg/kg12.50 mg/kg 0.252 0.5 mg/kg 25.00 mg/kg 0.361 0.5 mg/kg 50.00 mg/kg0.363

Example 29: Time Course of In Vivo, Antisense-Mediated Reduction ofMurine Factor 11 and Corresponding Anticoagulation in Blood Treatment

The time course of antisense-mediated reduction of murine Factor 11 mRNAwas observed in BALB/c mice. One dose of 50 mg/kg ISIS 404071 wasadministered subcutaneously to BALB/c mice. Following ISIS 404071administration, mice were sacrificed at 12 hours, 1 day, 2 days, 3 days,4 days, 7 days, 14 days, 28 days, and 56 days. Whole liver was collectedfor RNA analysis and PPP was collected for aPTT analysis. A controlgroup of mice was treated with one subcutaneous dose of PBS.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor11. Results are presented relative to PBS control. Mice treated withISIS 404071 showed significant Factor 11 mRNA down-regulation by day 1.Mice began regaining Factor 11 mRNA expression by day 14. Mice regainedfull Factor 11 mRNA expression by day 28 and results from day 56indicate that Factor mRNA was maintained at pre-treatment levels.Therefore, ISIS 404071 treated mice did not experience a rebound effect.

The rebound effect has been previously observed in antibody-mediatedreduction of Factor 11 (Blood, First Edition Paper, prepublished onlineOct. 22, 2008; Prevention of vascular graft occlusion andthrombus-associated thrombin generation by inhibition of factor XI).Because over expression of Factor 11 can be damaging by leading toincreased coagulation, these data suggest that antisense-mediatedinhibition of Factor 11 is safer than antibody-mediated inhibition ofFactor 11 since antisense-mediated inhibition of Factor 11 does notrebound.

aPTT Assay

aPTT values provided in Table 43 are reported as InternationalNormalized Ratio (INR) values. INR values for aPTT were determined bydividing the aPTT value for ISIS 404071 treated mice by the aPTT for thePBS treated group. This ratio was then raised to the power of theInternational Sensitivity Index (ISI) of the tissue factor used. Asshown in Table 43, mice treated with ISIS 404071 showed progressivedecrease of aPTT until day 4 and then progressive increase topre-treatment levels from day 7 to day 28.

TABLE 43 Effect of ISIS 404071 treatment on aPTT INR* 12 day day day dayday day day day hours 1 2 3 4 7 14 28 56 ISIS 404071 0 1.02 1.12 1.291.30 1.25 1.11 1.02 0 *values in Table 43 are approximate

Example 30: Antisense Inhibition of Human Factor 11 in HepG2 Cells byOligonucleotides Designed by Microwalk

Additional gapmers were designed based on ISIS 416850 and ISIS 416858(see Table 8 above). These gapmers were shifted slightly upstream anddownstream (i.e. “microwalk”) of ISIS 416850 and ISIS 416858. Themicrowalk gapmers were designed with either 5-8-5 MOE or 6-8-6 MOEmotifs.

These microwalk gapmers were tested in vitro. Cultured HepG2 cells at adensity of 20,000 cells per well were transfected using electroporationwith 8,000 nM antisense oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and Factor 11mRNA levels were measured by quantitative real-time PCR. Factor 11 mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN. Results are presented as percent inhibition of Factor 11,relative to untreated control cells.

ISIS 416850 and ISIS 416858, as well as selected gapmers from Tables 1and 8 (i.e., ISIS 412206, ISIS 412223, ISIS 412224, ISIS 412225, ISIS413481, ISIS 413482, ISIS 416825, ISIS 416848, ISIS 416849, ISIS 416850,ISIS 416851, ISIS 416852, ISIS 416853, ISIS 416854, ISIS 416855, ISIS416856, ISIS 416857, ISIS 416858, ISIS 416859, ISIS 416860, ISIS 416861,ISIS 416862, ISIS 416863, ISIS 416864, ISIS 416865, ISIS 416866, andISIS 416867) were retested in vitro along with the microwalk gapmersunder the same condition as described above.

The chimeric antisense oligonucleotides in Table 44 were designed as5-10-5 MOE, 5-8-5 and 6-8-6 MOE gapmers. The first two listed gapmers inTable 44 are the original gapmers (ISIS 416850 and ISIS 416858) fromwhich ISIS 445493-445543 were designed via microwalk, and are designatedby an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, whereinthe central gap segment is comprised of ten 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprisingfive nucleotides each. The 5-8-5 gapmers are 18 nucleotides in length,wherein the central gap segment is comprised of eight2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′directions) by wings comprising five nucleotides each. The 6-8-6 gapmersare 20 nucleotides in length, wherein the central gap segment iscomprised of eight 2′-deoxynucleotides and is flanked on both sides (inthe 5′ and 3′ directions) by wings comprising six nucleotides each. Foreach of the motifs (5-10-5, 5-8-5 and 6-8-6), each nucleotide in the 5′wing segment and each nucleotide in the 3′ wing segment has a 2′-MOEmodification. The internucleoside linkages throughout each gapmer arephosphorothioate (P=S) linkages. All cytidine residues throughout eachgapmer are 5-methylcytidines. “Human Target start site” indicates the5′-most nucleotide to which the gapmer is targeted in the humansequence. “Human Target stop site” indicates the 3′-most nucleotide towhich the gapmer is targeted in the human sequence. Each gapmer listedin Table 44 is targeted to SEQ ID NO: 1 (GENBANK Accession No.NM_000128.3). Each gapmer is Table 44 is also fully cross-reactive withthe rhesus monkey Factor 11 gene sequence, designated herein as SEQ IDNO: 274 (exons 1-15 GENBANK Accession No. NW_001118167.1). ‘Rhesusmonkey start site’ indicates the 5′-most nucleotide to which the gapmeris targeted in the rhesus monkey sequence. ‘Rhesus monkey stop site’indicates the 3′-most nucleotide to which the gapmer is targeted to therhesus monkey sequence.

As shown in Table 44, all of the microwalk designed gapmers targeted tothe target region beginning at the target start site 1275 and ending atthe target stop site 1317 (i.e. nucleobases 1275-1317) of SEQ ID NO: 1exhibited at least 60% inhibition of Factor 11 mRNA. Similarly, all ofthe re-tested gapmers from Tables 1 and 8 exhibited at least 60%inhibition.

Several of the gapmers exhibited at least 70% inhibition, including ISISnumbers: ISIS 412206, 412224, 412225, 413481, 413482, 416825, 416848,416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857,416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866,416867, 445494, 445495, 445496, 445497, 445498, 445499, 445500, 445501,445502, 445503, 445504, 445505, 445506, 445507, 445508, 445509, 445510,445511, 445512, 445513, 445514, 445515, 445516, 445517, 445518, 445519,445520, 445521, 445522, 445523, 445524, 445525, 445526, 445527, 445528,445529, 445530, 445531, 445532, 445533, 445534, 445535, 445536, 445537,455538, 445539, 445540, 445541, 445542, and 445543.

Several of the gapmers exhibited at least 80% inhibition, including ISISnumbers: ISIS 412206, 412224, 412225, 413481, 413482, 416825, 416848,416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857,416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866,416867, 445494, 445495, 445496, 445497, 445498, 445500, 445501, 445502,445503, 445504, 445505, 445506, 445507, 445508, 445509, 445510, 445513,445514, 445519, 445520, 445521, 445522, 445525, 445526, 445529, 445530,445531, 445532, 445533, 445534, 445535, 445536, 455538, 445541, and445542.

Several of the gapmers exhibited at least 90% inhibition, including ISISnumbers: ISIS 412206, 416825, 416850, 416857, 416858, 416861, 445522,and 445531.

TABLE 44Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted toSEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) Rhesus Rhesus HumanHuman SEQ monkey monkey Start Stop Sequence Percent ID Start StopISIS No. Site Site (5′ to 3′) inhibition Motif No. Site Site *4168501278 1297 TGCACAGTTTCT 91 5-10-5 215 1277 1296 GGCAGGCC *416858 12881307 ACGGCATTGGT 90 5-10-5 223 1287 1306 GCACAGTTT  416825 680 699GCCCTTCATGTC 90 5-10-5 190 679 698 TAGGTCCA  412206 738 757 CCGTGCATCTTT91 5-10-5 34 737 756 CTTGGCAT  412223 1275 1294 ACAGTTTCTGG 62 5-10-5 511274 1293 CAGGCCTCG  445493 1275 1294 ACAGTTTCTGG 69 6-8-6 51 1274 1293CAGGCCTCG  445518 1275 1292 AGTTTCTGGCA 75 5-8-5 242 1274 1291 GGCCTCG 416848 1276 1295 CACAGTTTCTG 87 5-10-5 213 1275 1294 GCAGGCCTC  4454941276 1295 CACAGTTTCTG 85 6-8-6 213 1275 1294 GCAGGCCTC  445519 1276 1293CAGTTTCTGGC 81 5-8-5 243 1275 1292 AGGCCTC  416849 1277 1296 GCACAGTTTCT88 5-10-5 214 1276 1295 GGCAGGCCT  445495 1277 1296 GCACAGTTTCT 89 6-8-6214 1276 1295 GGCAGGCCT  445520 1277 1294 ACAGTTTCTGG 82 5-8-5 244 12761293 CAGGCCT  445496 1278 1297 TGCACAGTTTCT 87 6-8-6 215 1277 1296GGCAGGCC  445521 1278 1295 CACAGTTTCTG 87 5-8-5 245 1277 1294 GCAGGCC 416851 1279 1298 GTGCACAGTTT 89 5-10-5 216 1278 1297 CTGGCAGGC  4454971279 1298 GTGCACAGTTT 81 6-8-6 216 1278 1297 CTGGCAGGC  445522 1279 1296GCACAGITTCT 91 5-8-5 246 1278 1295 GGCAGGC  413481 1280 1299 GGTGCACAGTT82 5-10-5 114 1279 1298 TCTGGCAGG  445498 1280 1299 GGTGCACAGTT 83 6-8-6114 1279 1298 TCTGGCAGG  445523 1280 1297 TGCACAGTTTCT 73 5-8-5 267 12791296 GGCAGG  416852 1281 1300 TGGTGCACAGT 87 5-10-5 217 1280 1299TTCTGGCAG  445499 1281 1300 TGGTGCACAGT 75 6-8-6 217 1280 1299 TTCTGGCAG 445524 1281 1298 GTGCACAGTTT 75 5-8-5 247 1280 1297 CTGGCAG  4168531282 1301 TTGGTGCACAG 84 5-10-5 218 1281 1300 TTTCTGGCA  445500 12821301 TTGGTGCACAG 81 6-8-6 218 1281 1300 TTTCTGGCA  445525 1282 1299GGTGCACAGTT 85 5-8-5 248 1281 1298 TCTGGCA  416854 1283 1302 ATTGGTGCACA86 5-10-5 219 1282 1301 GTTTCTGGC  445501 1283 1302 ATTGGTGCACA 83 6-8-6219 1282 1301 GTTTCTGGC  445526 1283 1300 TGGTGCACAGT 81 5-8-5 249 12821299 TTCTGGC  416855 1284 1303 CATTGGTGCAC 85 5-10-5 220 1283 1302AGTTTCTGG  445502 1284 1303 CATTGGTGCAC 83 6-8-6 220 1283 1302 AGTTTCTGG 445527 1284 1301 TTGGTGCACAG 70 5-8-5 250 1283 1300 TTTCTGG  4122241285 1304 GCATTGGTGCA 84 5-10-5 52 1284 1303 CAGTTTCTG  445503 1285 1304GCATTGGTGCA 89 6-8-6 52 1284 1303 CAGTTTCTG  445528 1285 1302ATTGGTGCACA 73 5-8-5 251 1284 1301 GTTTCTG  416856 1286 1305 GGCATTGGTGC84 5-10-5 221 1285 1304 ACAGTTTCT  445504 1286 1305 GGCATTGGTGC 87 6-8-6221 1285 1304 ACAGTTTCT  445529 1286 1303 CATTGGTGCAC 85 5-8-5 252 12851302 AGTTTCT  416857 1287 1306 CGGCATTGGTG 91 5-10-5 222 1286 1305CACAGTTTC  445505 1287 1306 CGGCATTGGTG 89 6-8-6 222 1286 1305 CACAGTTTC 445530 1287 1304 GCATTGGTGCA 83 5-8-5 253 1286 1303 CAGTTTC  4455061288 1307 ACGGCATTGGT 86 6-8-6 223 1287 1306 GCACAGTTT  445531 1288 1305GGCATTGGTGC 90 5-8-5 254 1287 1304 ACAGTTT  416859 1289 1308 GACGGCATTGG85 5-10-5 224 1288 1307 TGCACAGTT  445507 1289 1308 GACGGCATTGG 85 6-8-6224 1288 1307 TGCACAGTT  445532 1289 1306 CGGCATTGGTG 89 5-8-5 255 12881305 CACAGTT  413482 1290 1309 GGACGGCATTG 88 5-10-5 115 1289 1308GTGCACAGT  445508 1290 1309 GGACGGCATTG 81 6-8-6 115 1289 1308 GTGCACAGT 445533 1290 1307 ACGGCATTGGT 87 5-8-5 256 1289 1306 GCACAGT  4168601291 1310 CGGACGGCATT 89 5-10-5 225 1290 1309 GGTGCACAG  445509 12911310 CGGACGGCATT 84 6-8-6 225 1290 1309 GGTGCACAG  445534 1291 1308GACGGCATTGG 82 5-8-5 257 1290 1307 TGCACAG  416861 1292 1311 GCGGACGGCAT90 5-10-5 226 1291 1310 TGGTGCACA  445510 1292 1311 GCGGACGGCAT 88 6-8-6226 1291 1310 TGGTGCACA  445535 1292 1309 GGACGGCATTG 83 5-8-5 258 12911308 GTGCACA  416862 1293 1312 AGCGGACGGCA 89 5-10-5 227 1292 1311TTGGTGCAC  445511 1293 1312 AGCGGACGGCA 77 6-8-6 227 1292 1311 TTGGTGCAC 445536 1293 1310 CGGACGGCATT 82 5-8-5 259 1292 1309 GGTGCAC  4168631294 1313 CAGCGGACGGC 86 5-10-5 228 1293 1312 ATTGGTGCA  445512 12941313 CAGCGGACGGC 79 6-8-6 228 1293 1312 ATTGGTGCA  445537 1294 1311GCGGACGGCAT 78 5-8-5 260 1293 1310 TGGTGCA  412225 1295 1314 GCAGCGGACGG86 5-10-5 53 1294 1313 CATTGGTGC  445513 1295 1314 GCAGCGGACGG 85 6-8-653 1294 1313 CATTGGTGC  445538 1295 1312 AGCGGACGGCA 80 5-8-5 261 12941311 TTGGTGC  416864 1296 1315 GGCAGCGGACG 88 5-10-5 229 1295 1314GCATTGGTG  445514 1296 1315 GGCAGCGGACG 81 6-8-6 229 1295 1314 GCATTGGTG 445539 1296 1313 CAGCGGACGGC 79 5-8-5 262 1295 1312 ATTGGTG  4168651297 1316 TGGCAGCGGAC 86 5-10-5 230 1296 1315 GGCATTGGT  445515 12971316 TGGCAGCGGAC 75 6-8-6 230 1296 1315 GGCATTGGT  445540 1297 1314GCAGCGGACGG 74 5-8-5 263 1296 1313 CATTGGT  416866 1298 1317 CTGGCAGCGGA84 5-10-5 231 1297 1316 CGGCATTGG  445516 1298 1317 CTGGCAGCGGA 79 6-8-6231 1297 1316 CGGCATTGG  445541 1298 1315 GGCAGCGGACG 80 5-8-5 264 12971314 GCATTGG  416867 1299 1318 ACTGGCAGCGG 85 5-10-5 232 1298 1317ACGGCATTG  445517 1299 1318 ACTGGCAGCGG 74 6-8-6 232 1298 1317 ACGGCATTG 445542 1299 1316 TGGCAGCGGAC 83 5-8-5 265 1298 1315 GGCATTG  4455431300 1317 CTGGCAGCGGA 74 5-8-5 266 1299 1316 CGGCATT

Example 31: Dose-Dependent Antisense Inhibition of Human Factor 11 inHepG2 Cells

Gapmers from Example 30 exhibiting in vitro inhibition of human Factor11 were tested at various doses in HepG2 cells. Cells were plated at adensity of 20,000 cells per well and transfected using electroporationwith 123.46 nM, 370.37 nM, 1,111.11 nM, 3,333.33 nM and 10,000 nMconcentrations of antisense oligonucleotide, as specified in Table 45.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and Factor 11 mRNA levels were measured by quantitativereal-time PCR. Human Factor 11 primer probe set RTS 2966 was used tomeasure mRNA levels. Factor 11 mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN. Results are presented aspercent inhibition of Factor 11, relative to untreated control cells. Asillustrated in Table 45, Factor 11 mRNA levels were reduced in adose-dependent manner in antisense oligonucleotide treated cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotidewas calculated by plotting the concentrations of antisenseoligonucleotides used versus the percent inhibition of Factor 11 mRNAexpression achieved at each concentration, and noting the concentrationof antisense oligonucleotide at which 50% inhibition of Factor 11 mRNAexpression was achieved compared to the PBS control. IC₅₀ values arepresented in Table 45.

TABLE 45 Dose-dependent antisense inhibition of human Factor 11 in HepG2cells via transfection of oligonucleotides using electroporation ISIS123.47 370.37 1,111.11 3,333.33 10,000.0 IC₅₀ No. nM nM nM nM nM (μM)416849 5 5 26 57 68 2.7 416850 0 12 36 74 73 2.8 416851 13 35 36 64 721.5 416856 12 23 35 59 83 1.6 416857 2 20 35 62 72 2.3 416858 0 27 36 6470 2.2 416860 0 28 39 41 40 n.d. 416861 0 15 27 66 80 2.0 445498 3 1 2750 58 4.8 445503 0 0 22 36 60 5.9 445504 8 20 38 53 68 2.7 445505 12 3039 59 77 1.8 445522 0 0 44 63 74 2.9 445531 8 16 52 61 77 1.8 445532 512 39 60 70 2.0 n.d. = no data

Example 32: Dose-Dependent Antisense Inhibition of Human Factor 11 inHepG2 Cells by Oligonucleotides Designed by Microwalk

Additional gapmers were designed based on ISIS 416850 and ISIS 416858(see Table 8 above). These gapmers are shifted slightly upstream anddownstream (i.e. microwalk) of ISIS 416850 and ISIS 416858. Gapmersdesigned by microwalk have 3-8-3 MOE, 4-8-4 MOE, 2-10-2 MOE, 3-10-3 MOE,or 4-10-4 MOE motifs.

These gapmers were tested at various doses in HepG2 cells. Cells wereplated at a density of 20,000 cells per well and transfected usingelectroporation with 375 nM, 750 nM, 1,500 nM, 3,000 nM, 6,000 nM and12,000 nM concentrations of antisense oligonucleotide, as specified inTable 47. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and Factor 11 mRNA levels were measured byquantitative real-time PCR. Human Factor 11 primer probe set RTS 2966was used to measure mRNA levels. Factor 11 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN. Results arepresented as percent inhibition of Factor 11, relative to untreatedcontrol cells.

ISIS 416850, ISIS 416858, ISIS 445522, and ISIS 445531 (see Table 45above) were re-tested in vitro along with the microwalk gapmers underthe same conditions described above.

The chimeric antisense oligonucleotides in Table 46 were designed as3-8-3 MOE, 4-8-4 MOE, 2-10-2 MOE, 3-10-3 MOE, or 4-10-4 MOE gapmers. The3-8-3 gapmer is 14 nucleotides in length, wherein the central gapsegment is comprised of eight 2′-deoxynucleotides and is flanked on bothsides (in the 5′ and 3′ directions) by wings comprising threenucleotides each. The 4-8-4 gapmer is 16 nucleotides in length, whereinthe central gap segment is comprised of eight 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprisingfour nucleotides each. The 2-10-2 gapmer is 14 nucleotides in length,wherein the central gap segment is comprised of ten 2′-deoxynucleotidesand is flanked on both sides (in the 5′ and 3′ directions) by wingscomprising two nucleotides each. The 3-10-3 gapmer is 16 nucleotides inlength, wherein the central gap segment is comprised of ten2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′directions) by wings comprising three nucleotides each. The 4-10-4gapmer is 18 nucleotides in length, wherein the central gap segment iscomprised of ten 2′-deoxynucleotides and is flanked on both sides (inthe 5′ and 3′ directions) by wings comprising four nucleotides each. Foreach of the motifs (3-8-3, 4-8-4, 2-10-2, 3-10-3, and 4-10-4), eachnucleotide in the 5′ wing segment and each nucleotide in the 3′ wingsegment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P=S) linkages. All cytidineresidues throughout each gapmer are 5-methylcytidines. “Human Targetstart site” indicates the 5′-most nucleotide to which the gapmer istargeted in the human sequence. “Human Target stop site” indicates the3′-most nucleotide to which the gapmer is targeted in the humansequence. Each gapmer listed in Table 46 is targeted to SEQ ID NO: 1(GENBANK Accession No. NM_000128.3). Each gapmer is Table 46 is alsofully cross-reactive with the rhesus monkey Factor 11 gene sequence,designated herein as SEQ ID NO: 274 (exons 1-15 GENBANK Accession No.NW_001118167.1). ‘Rhesus monkey start site’ indicates the 5′-mostnucleotide to which the gapmer is targeted in the rhesus monkeysequence. ‘Rhesus monkey stop site’ indicates the 3′-most nucleotide towhich the gapmer is targeted to the rhesus monkey sequence.

TABLE 46Chimeric antisense oligonucleotides targeted to SEQ ID NO: 1 (GENBANK Accession No.NM_000128.3) and designed by microwalk of ISIS 416850 and ISIS 416858Human Human Rhesus Rhesus Target Target SEQ monkey monkey ISIS StartStop ID Start Stop No. Site Site Sequence (5′ to 3′) Motif No. Site Site449707 1280 1295 CACAGTTT 4-8-4 268 1279 1294 CTGGCAGG 449708 1281 1294ACAGTTT 3-8-3 269 1280 1293 CTGGCAG 449709 1279 1296 GCACAGTT 4-10-4 2461278 1295 TCTGGCAGGC 449710 1280 1295 CACAGTTT 3-10-3 268 1279 1294CTGGCAGG 449711 1281 1294 ACAGTTT 2-10-2 269 1280 1293 CTGGCAG

Dose-response inhibition data is given in Table 47. As illustrated inTable 47, Factor 11 mRNA levels were reduced in a dose-dependent mannerin antisense oligonucleotide treated cells. The IC₅₀ of each antisenseoligonucleotide was also calculated and presented in Table 47. The firsttwo listed gapmers in Table 47 are the original gapmers (ISIS 416850 andISIS 416858) from which the remaining gapmers were designed viamicrowalk and are designated by an asterisk.

TABLE 47 Dose-dependent antisense inhibition of human Factor 11 in HepG2cells via transfection of oligonucleotides using electroporation ISIS375 750 1,500 3,000 6,000 12,000 IC₅₀ No. nM nM nM nM nM nM (μM)*416850  40 59 69 87 90 95 0.56 *416858  31 35 78 85 90 93 0.83 44552259 71 83 82 81 92 n.d. 445531 44 64 78 86 91 93 0.44 449707 7 35 63 7385 91 1.26 449708 0 0 22 33 61 85 4.46 449709 52 71 80 87 92 95 0.38449710 2 21 52 70 82 87 1.59 449711 6 14 1 7 32 52 11.04 n.d. = no data

Example 33: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in CD1 Mice

CD1 mice were treated with ISIS antisense oligonucleotides targetinghuman Factor 11 and evaluated for changes in the levels of variousmetabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice a weekfor 2, 4, or 6 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925,ISIS 416999, ISIS 417002, or ISIS 417003. A control group of five micewas injected subcutaneously with PBS for 2 weeks. All experimentalgroups (i.e. ASO treated mice at 2, 4, 6 weeks) were compared to thecontrol group (i.e. PBS, 2 weeks).

Three days after the last dose was administered to all groups, the micewere sacrificed. Organ weights were measured and blood was collected forfurther analysis.

Organ Weight

Liver, spleen, and kidney weights were measured at the end of the study,and are presented in Tables 48, 49, and 50 as a percent of the PBScontrol, normalized to body weight. Those antisense oligonucleotideswhich did not affect more than six-fold increases in liver and spleenweight above the PBS controls were selected for further studies.

TABLE 48 Percent change in liver weight of CD1 mice after antisenseoligonucleotide treatment ISIS No. 2 weeks 4 weeks 6 weeks 416825 +5 +22+13 416826 +10 +32 +33 416838 +8 −6 0 416850 +5 +3 +6 416858 +7 +1 +10416864 −2 +2 −5 416925 +14 +14 +33 416999 +13 +30 +47 417002 +14 +8 +35416892 +35 +88 +95 417003 +8 +42 +32

TABLE 49 Percent change in spleen weight of CD1 mice after antisenseoligonucleotide treatment ISIS No. 2 weeks 4 weeks 6 weeks 416825 −12+19 +21 416826 −12 −5 +22 416838 +21 −8 +9 416850 −4 +6 +48 416858 −2 +8+28 416864 −10 −2 −6 416925 −7 +33 +78 416999 +7 +22 +38 417002 +29 +26+108 416892 +24 +30 +65 417003 +12 +101 +98

TABLE 50 Percent change in kidney weight of CD1 mice after antisenseoligonucleotide treatment ISIS No. 2 weeks 4 weeks 6 weeks 416825 −12−12 −11 416826 −13 −7 −22 416838 −2 −12 −8 416850 −0 −12 −11 416858 +1−18 −10 416864 −4 −9 −15 416925 −4 −14 −2 416999 −9 −6 −7 417002 +3 −5−2 416892 +2 −3 +19 417003 −9 −2 −1

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Measurements of alanine transaminase (ALT) and aspartate transaminase(AST) are expressed in IU/L and the results are presented in Tables 51and 52. Plasma levels of bilirubin and albumin were also measured usingthe same clinical chemistry analyzer and expressed in mg/dL. The resultsare presented in Tables 53 and 54. Those antisense oligonucleotideswhich did not affect an increase in ALT/AST levels above seven-fold ofcontrol levels were selected for further studies. Those antisenseoligonucleotides which did not increase levels of bilirubin more thantwo-fold of the control levels were selected for further studies.

TABLE 51 Effect of antisense oligonucleotide treatment on ALT (IU/L) inCD1 mice 2 weeks 4 weeks 6 weeks PBS 36 n.d. n.d. ISIS 416825 64 314 507ISIS 416826 182 126 1954 ISIS 416838 61 41 141 ISIS 416850 67 58 102ISIS 416858 190 57 216 ISIS 416864 44 33 92 ISIS 416925 160 284 1284ISIS 416999 61 160 1302 ISIS 417002 71 138 2579 ISIS 416892 66 1526 1939ISIS 417003 192 362 2214 n.d. = no data

TABLE 52 Effect of antisense oligonucleotide treatment on AST (IU/L) inCD1 mice 2 weeks 4 weeks 6 weeks PBS 68 n.d. n.d. ISIS 416825 82 239 301ISIS 416826 274 156 1411 ISIS 416838 106 73 107 ISIS 416850 72 88 97ISIS 416858 236 108 178 ISIS 416864 58 46 101 ISIS 416925 144 206 712ISIS 416999 113 130 671 ISIS 417002 96 87 1166 ISIS 416892 121 1347 1443ISIS 417003 152 249 839 n.d. = no data

TABLE 53 Effect of antisense oligonucleotide treatment on bilirubin(mg/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 0.28 n.d. n.d. ISIS416825 0.41 0.69 0.29 ISIS 416826 0.39 0.20 0.37 ISIS 416838 0.57 0.240.20 ISIS 416850 0.46 0.23 0.22 ISIS 416858 0.57 0.24 0.16 ISIS 4168640.40 0.26 0.22 ISIS 416925 0.45 0.25 0.25 ISIS 416999 0.48 0.18 0.28ISIS 417002 0.50 0.25 0.29 ISIS 416892 0.38 2.99 0.50 ISIS 417003 0.330.15 0.24 n.d. = no data

TABLE 54 Effect of antisense oligonucleotide treatment on albumin(mg/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 3.7 n.d. n.d. ISIS416825 3.6 3.4 3.5 ISIS 416826 3.3 3.4 3.4 ISIS 416838 3.5 3.8 3.6 ISIS416850 3.6 3.5 3.1 ISIS 416858 3.4 3.5 2.8 ISIS 416864 3.5 3.6 3.5 ISIS416925 3.5 3.5 3.2 ISIS 416999 3.4 3.3 3.2 ISIS 417002 3.2 3.4 3.4 ISIS416892 3.2 4.0 4.4 ISIS 417003 3.4 3.4 3.2 n.d. = no data

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.). Results are presented in Tables 55 and 56,expressed in mg/dL. Those antisense oligonucleotides which did notaffect more than a two-fold increase in BUN levels compared to the PBScontrol were selected for further studies.

TABLE 55 Effect of antisense oligonucleotide treatment on BUN (mg/dL) inCD1 mice 2 weeks 4 weeks 6 weeks PBS 30 n.d. n.d. ISIS 416825 29 35 31ISIS 416826 24 34 27 ISIS 416838 25 38 30 ISIS 416850 25 30 23 ISIS416858 21 29 19 ISIS 416864 22 31 28 ISIS 416925 21 30 17 ISIS 416999 2227 22 ISIS 417002 19 23 19 ISIS 416892 19 28 23 ISIS 417003 23 26 24n.d. = no data

TABLE 56 Effect of antisense oligonucleotide treatment on creatinine(mg/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 0.14 n.d. n.d. ISIS416825 0.14 0.21 0.17 ISIS 416826 0.15 0.20 0.15 ISIS 416838 0.09 0.270.14 ISIS 416850 0.13 0.22 0.19 ISIS 416858 0.13 0.23 0.10 ISIS 4168640.11 0.22 0.16 ISIS 416925 0.12 0.25 0.13 ISIS 416999 0.07 0.18 0.13ISIS 417002 0.06 0.16 0.10 ISIS 416892 0.11 0.20 0.17 ISIS 417003 0.170.24 0.18 n.d. = no data

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics forhematocrit (HCT), mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)measurements and analyses, as well as measurements of the various bloodcells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, andplatelets, and total hemoglobin content. The results are presented inTables 57-67. Percentages given in the tables indicate the percent oftotal blood cell count. Those antisense oligonucleotides which did notaffect a decrease in platelet count of more than 50% and/or an increasein monocyte count of more than three-fold were selected for furtherstudies.

TABLE 57 Effect of antisense oligonucleotide treatment on HCT (%) in CD1mice 2 weeks 4 weeks 6 weeks PBS 50 n.d. n.d. ISIS 416825 49 46 40 ISIS416826 47 41 37 ISIS 416838 42 44 39 ISIS 416850 44 44 38 ISIS 416858 5045 46 ISIS 416864 50 45 42 ISIS 416925 51 47 47 ISIS 416999 51 42 40ISIS 417002 44 44 51 ISIS 416892 48 42 45 ISIS 417003 48 41 43 n.d. = nodata

TABLE 58 Effect of antisense oligonucleotide treatment on MCV (fL) inCD1 mice 2 weeks 4 weeks 6 weeks PBS 61 n.d. n.d. ISIS 416825 58 53 51ISIS 416826 56 52 53 ISIS 416838 56 54 48 ISIS 416850 57 51 50 ISIS416858 59 51 50 ISIS 416864 57 52 51 ISIS 416925 61 52 47 ISIS 416999 6049 48 ISIS 417002 61 50 52 ISIS 416892 59 49 53 ISIS 417003 60 48 45n.d. = no data

TABLE 59 Effect of antisense oligonucleotide treatment on MCH (pg) inCD1 mice ISIS No. 2 weeks 4 weeks 6 weeks PBS 18 n.d. n.d. ISIS 41682517 16 15 ISIS 416826 17 16 16 ISIS 416838 17 17 15 ISIS 416850 17 16 15ISIS 416858 17 16 15 ISIS 416864 18 16 16 ISIS 416925 17 16 15 ISIS416999 17 16 15 ISIS 417002 17 16 16 ISIS 416892 18 16 16 ISIS 417003 1716 16 n.d. = no data

TABLE 60 Effect of antisense oligonucleotide treatment on MCHC (%) inCD1 mice 2 weeks 4 weeks 6 weeks PBS 30 n.d. n.d. ISIS 416825 29 31 31ISIS 416826 29 31 30 ISIS 416838 30 31 32 ISIS 416850 30 31 31 ISIS416858 30 32 31 ISIS 416864 31 31 31 ISIS 416925 30 32 32 ISIS 416999 2732 31 ISIS 417002 29 32 31 ISIS 416892 30 32 30 ISIS 417003 29 32 33n.d. = no data

TABLE 61 Effect of antisense oligonucleotide treatment on WBC count(cells/nL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 6 n.d. n.d. ISIS416825 8 8 6 ISIS 416826 5 6 8 ISIS 416838 4 6 5 ISIS 416850 4 5 5 ISIS416858 6 7 4 ISIS 416864 7 6 5 ISIS 416925 6 6 11  ISIS 416999 4 9 7ISIS 417002 8 8 16  ISIS 416892 5 8 9 ISIS 417003 7 9 10  n.d. = no data

TABLE 62 Effect of antisense oligonucleotide treatment on RBC count(cells/pL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 8 n.d. n.d. ISIS416825 9 9 8 ISIS 416826 8 8 7 ISIS 416838 8 8 8 ISIS 416850 8 9 8 ISIS416858 9 9 9 ISIS 416864 9 9 8 ISIS 416925 9 9 10  ISIS 416999 9 9 8ISIS 417002 9 9 10  ISIS 416892 7 9 9 ISIS 417003 8 9 10  n.d. = no data

TABLE 63 Effect of antisense oligonucleotide treatment on neutrophilcount (%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 16 n.d. n.d. ISIS416825 15 43 23 ISIS 416826 26 33 23 ISIS 416838 19 33 31 ISIS 416850 1521 16 ISIS 416858 14 24 27 ISIS 416864 13 27 20 ISIS 416925 12 39 33ISIS 416999 12 25 22 ISIS 417002 14 31 36 ISIS 416892 19 43 28 ISIS417003 10 39 24 n.d. = no data

TABLE 64 Effect of antisense oligonucleotide treatment on lymphocytecount (%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 81 n.d. n.d. ISIS416825 82 53 71 ISIS 416826 70 61 67 ISIS 416838 76 64 60 ISIS 416850 8273 76 ISIS 416858 83 73 65 ISIS 416864 84 71 74 ISIS 416925 86 58 57ISIS 416999 86 72 69 ISIS 417002 83 64 51 ISIS 416892 79 52 64 ISIS417003 86 54 66 n.d. = no data

TABLE 65 Effect of antisense oligonucleotide treatment on monocyte count(%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 3 n.d. n.d. ISIS 416825 2 54 ISIS 416826 3 5 8 ISIS 416838 2 2 6 ISIS 416850 3 6 6 ISIS 416858 2 37 ISIS 416864 2 2 5 ISIS 416925 2 4 8 ISIS 416999 2 4 8 ISIS 417002 3 412  ISIS 416892 3 6 7 ISIS 417003 2 6 8 n.d. = no data

TABLE 66 Effect of antisense oligonucleotide treatment on platelet count(cells/nL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 2126 n.d. n.d. ISIS416825 1689 1229 942 ISIS 416826 1498 970 645 ISIS 416838 1376 1547 1229ISIS 416850 1264 1302 1211 ISIS 416858 2480 1364 1371 ISIS 416864 19241556 933 ISIS 416925 1509 1359 1211 ISIS 416999 1621 1219 1057 ISIS417002 1864 1245 1211 ISIS 416892 1687 636 1004 ISIS 417003 1309 773 922n.d. = no data

TABLE 67 Effect of antisense oligonucleotide treatment on hemoglobincontent (g/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 15.1 n.d. n.d.ISIS 416825 14.5 14.1 12.1 ISIS 416826 13.4 12.8 11.0 ISIS 416838 12.413.6 12.6 ISIS 416850 13.1 13.5 11.6 ISIS 416858 14.8 14.2 14.1 ISIS416864 15.2 13.9 13.0 ISIS 416925 14.9 14.8 15.3 ISIS 416999 14.2 13.312.8 ISIS 417002 14.7 14.0 15.7 ISIS 416892 13.0 13.5 13.1 ISIS 41700313.7 13.4 14.0 n.d. = no data

Example 34: Measurement of Half-Life of Antisense Oligonucleotide in CD1Mice Liver

CD1 mice were treated with ISIS antisense oligonucleotides targetinghuman Factor 11 and the oligonucleotide half-life as well as the elapsedtime for oligonucleotide degradation and elimination from the liver wasevaluated.

Treatment

Groups of fifteen CD1 mice each were injected subcutaneously twice perweek for 2 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS 416838,ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925, ISIS416999, ISIS 417002, or ISIS 417003. Five mice from each group weresacrificed 3 days, 28 days and 56 days following the final dose. Liverswere harvested for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. Half-lives were then calculated using WinNonlinsoftware (PHARSIGHT).

The results are presented in Tables 68 and 69, expressed as μg/g livertissue. The half-life of each oligonucleotide is presented in Table 70.

TABLE 68 Full-length oligonucleotide concentration (μg/g) in the liverof CD1 mice ISIS No. Motif day 3 day 28 day 56 416825 5-10-5 151 52 7416826 5-10-5 186 48 8 416838 5-10-5 170 46 10 416850 5-10-5 238 93 51416858 5-10-5 199 102 18 416864 5-10-5 146 38 25 416999 2-13-5 175 26 0417002 2-13-5 119 24 1 417003 2-13-5 245 42 4 416925 3-14-3 167 39 5416892 3-14-3 135 31 6

TABLE 69 Total oligonucleotide concentration (μg/g) in the liver of CD1mice ISIS No. Motif day 3 day 28 day 56 416825 5-10-5 187 90 39 4168265-10-5 212 61 12 416838 5-10-5 216 98 56 416850 5-10-5 295 157 143416858 5-10-5 273 185 56 416864 5-10-5 216 86 112 416999 2-13-5 232 51 0417002 2-13-5 206 36 1 417003 2-13-5 353 74 4 416925 3-14-3 280 72 8416892 3-14-3 195 54 6

TABLE 70 Half-life of antisense oligonucleotides in the liver of CD1mice Half-life ISIS No. Motif (days) 416825 5-10-5 16 416826 5-10-5 13416838 5-10-5 13 416850 5-10-5 18 416858 5-10-5 26 416864 5-10-5 13416999 2-13-5 9 417002 2-13-5 11 417003 2-13-5 10 416925 3-14-3 12416892 3-14-3 12

Example 35: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotidestargeting human Factor 11 and evaluated for changes in the levels ofvarious metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice per week for 6 weeks with 50 mg/kg of ISIS 416825, ISIS 416826,ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416848, ISIS 416864, ISIS416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. A controlgroup of four Sprague Dawley rats was injected subcutaneously with PBStwice per week for 6 weeks. Body weight measurements were taken beforeand throughout the treatment period. Urine samples were taken before thestart of treatment. Three days after the last dose, urine samples weretaken and the rats were sacrificed. Organ weights were measured andblood was collected for further analysis.

Body Weight and Organ Weight

Body weights of the rats were measured at the onset of the study andsubsequently twice per week. The body weights are presented in Table 71and are expressed as a percent change over the weights taken at thestart of the study. Liver, spleen, and kidney weights were measured atthe end of the study and are presented in Table 71 as a percent of thesaline control normalized to body weight. Those antisenseoligonucleotides which did not affect more than a six-fold increase inliver and spleen weight above the PBS control were selected for furtherstudies.

TABLE 71 Percent change in organ weight of Sprague Dawley rats afterantisense oligonucleotide treatment ISIS Body No. Liver Spleen Kidneyweight 416825 +20 +245 +25 −18 416826 +81 +537 +44 −40 416838 +8 +212−0.5 −23 416850 +23 +354 +47 −33 416858 +8 +187 +5 −21 416864 +16 +204+16 −24 416925 +44 +371 +48 −32 416999 +51 +405 +71 −37 417002 +27 +446+63 −29 416892 +38 +151 +32 −39 417003 +51 +522 +25 −40

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Measurements of alanine transaminase (ALT) and aspartate transaminase(AST) are expressed in IU/L and the results are presented in Table 72.Those antisense oligonucleotides which did not affect an increase inALT/AST levels above seven-fold of control levels were selected forfurther studies. Plasma levels of bilirubin and albumin were alsomeasured with the same clinical analyzer and the results are alsopresented in Table 72, expressed in mg/dL. Those antisenseoligonucleotides which did not affect an increase in levels of bilirubinmore than two-fold of the control levels by antisense oligonucleotidetreatment were selected for further studies.

TABLE 72 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin(IU/L) (IU/L) (mg/dL) (mg/dL) PBS 9 5 20 2 ISIS 416825 89 17 4 2 ISIS416826 611 104 115 6 ISIS 416838 5 2 4 2 ISIS 416850 80 5 1 4 ISIS416858 13 4 4 2 ISIS 416864 471 68 3 4 ISIS 416925 102 20 13 5 ISIS416999 92 28 54 5 ISIS 417002 44 11 12 3 ISIS 416892 113 183 1 8 ISIS417003 138 23 50 6

Kidney Function

To evaluate the effect of kidney function, plasma concentrations ofblood urea nitrogen (BUN) and creatinine were measured using anautomated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,N.Y.). Results are presented in Table 73, expressed in mg/dL. Thoseantisense oligonucleotides which did not affect more than a two-foldincrease in BUN levels compared to the PBS control were selected forfurther studies. The ratio of urine protein to creatinine in total urinesamples was also calculated before and after antisense oligonucleotidetreatment and is presented in Table 74. Those antisense oligonucleotideswhich did not affect more than a five-fold increase in urineprotein/creatinine ratios compared to the PBS control were selected forfurther studies.

TABLE 73 Effect of antisense oligonucleotide treatment on metabolicmarkers in the kidney of Sprague-Dawley rats BUN Creatinine PBS 4 8 ISIS416825 7 17 ISIS 416826 25 6 ISIS 416838 4 5 ISIS 416850 5 7 ISIS 4168588 4 ISIS 416864 5 6 ISIS 416925 7 5 ISIS 416999 2 4 ISIS 417002 11 1ISIS 416892 188 1 ISIS 417003 9 9

TABLE 74 Effect of antisense oligonucleotide treatment on urine protein/creatinine ratio in Sprague Dawley rats Before After PBS 1.2 1.3 4168251.1 5.4 416826 1.0 11.4 416838 1.2 3.7 416850 1.0 4.0 416858 0.9 4.4416864 1.2 4.0 416925 1.0 4.3 416999 1.3 9.1 417002 1.0 2.4 416892 0.821.3 417003 0.9 4.8

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics forhematocrit (HCT), mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCV), and mean corpuscular hemoglobin concentration (MCHC)measurements and analyses, as well as measurements of various bloodcells, such as WBC (neutrophils, lymphocytes and monocytes), RBC, andplatelets as well as hemoglobin content. The results are presented inTables 75 and 76. Those antisense oligonucleotides which did not affecta decrease in platelet count of more than 50% and an increase inmonocyte count of more than three-fold were selected for furtherstudies.

TABLE 75 Effect of antisense oligonucleotide treatment on blood cellcount in Sprague-Dawley rats WBC RBC Neutrophils Lymphocytes MonocytesPlatelets (/nL) (/pL) (%) (%) (%) (10³/μL) PBS 21 6 37 7 26 18 ISIS416825 22 2 25 3 15 6 ISIS 416826 7 5 30 5 7 11 ISIS 416838 13 4 17 3 627 ISIS 416850 16 7 48 8 11 26 ISIS 416858 28 2 20 3 10 19 ISIS 41686415 4 26 2 29 12 ISIS 416925 24 6 20 4 23 8 ISIS 416999 12 5 23 3 20 12ISIS 417002 23 5 22 4 25 7 ISIS 416892 68 12 92 18 58 66 ISIS 417003 8311 17 3 6 19

TABLE 76 Effect of antisense oligonucleotide treatment on hematologicfactors (% control) in Sprague-Dawley rats Hemoglobin HCT MCV MCH MCHC(g/dL) (%) (fL) (pg) (%) PBS 6 4 6 2 4 ISIS 416825 2 2 4 2 4 ISIS 4168267 7 6 3 4 ISIS 416838 2 5 4 2 5 ISIS 416850 4 5 3 4 2 ISIS 416858 2 3 22 1 ISIS 416864 4 2 4 2 4 ISIS 416925 6 8 5 2 4 ISIS 416999 6 5 2 3 1ISIS 417002 5 7 7 3 5 ISIS 416892 14 13 1 2 0 ISIS 417003 11 8 6 4 4

Example 36: Measurement of Half-Life of Antisense Oligonucleotide inSprague-Dawley Rat Liver and Kidney

Sprague Dawley rats were treated with ISIS antisense oligonucleotidestargeting human Factor 11 and the oligonucleotide half-life as well asthe elapsed time for oligonucleotide degradation and elimination fromthe liver and kidney was evaluated.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice a week for 2 weeks with 20 mg/kg of ISIS416825, ISIS 416826, ISIS416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925,ISIS 416999, ISIS 417002, or ISIS 417003. Three days after the lastdose, the rats were sacrificed and livers and kidneys were collected foranalysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. The results are presented in Tables 77 and 78,expressed as μg/g liver or kidney tissue. Half-lives were thencalculated using WinNonlin software (PHARSIGHT).

TABLE 77 Full-length oligonucleotide concentration (μg/g) in the liverand kidney of Sprague-Dawley rats ISIS No. Motif Kidney Liver 4168255-10-5 632 236 416826 5-10-5 641 178 416838 5-10-5 439 171 416850 5-10-5259 292 416858 5-10-5 575 255 416864 5-10-5 317 130 416999 2-13-5 358267 417002 2-13-5 291 118 417003 2-13-5 355 199 416925 3-14-3 318 165416892 3-14-3 351 215

TABLE 78 Total oligonucleotide concentration (μg/g) in the liver andkidney of Sprague-Dawley rats ISIS No. Motif Kidney Liver 416825 5-10-5845 278 416826 5-10-5 775 214 416838 5-10-5 623 207 416850 5-10-5 352346 416858 5-10-5 818 308 416864 5-10-5 516 209 416999 2-13-5 524 329417002 2-13-5 490 183 417003 2-13-5 504 248 416925 3-14-3 642 267 4168923-14-3 608 316

TABLE 79 Half-life (days) of ISIS oligonucleotides in the liver andkidney of Sprague-Dawley rats ISIS No. Motif Half-life 416825 5-10-5 16416826 5-10-5 13 416838 5-10-5 13 416850 5-10-5 18 416858 5-10-5 26416864 5-10-5 13 416999 2-13-5 9 417002 2-13-5 11 417003 2-13-5 10416925 3-14-3 12 416892 3-14-3 12

Example 37: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in CD1 Mice

CD1 mice were treated with ISIS antisense oligonucleotides targetinghuman Factor 11 and evaluated for changes in the levels of variousmetabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice per weekfor 6 weeks with 50 mg/kg of ISIS 412223, ISIS 412224, ISIS 412225, ISIS413481, ISIS 413482, ISIS 416848, ISIS 416849, ISIS 416850, ISIS 416851,ISIS 416852, ISIS 416853, ISIS 416854, ISIS 416855, ISIS 416856, ISIS416857, ISIS 416858, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416862,ISIS 416863, ISIS 416864, ISIS 416865, ISIS 416866, or ISIS 416867, or.A control group of ten CD1 mice was injected subcutaneously with PBStwice per week for 6 weeks. Body weight measurements were taken beforeand throughout the treatment period. Three days after the last dose, themice were sacrificed, organ weights were measured, and blood wascollected for further analysis.

Body Weight and Organ Weights

Body weight was measured at the onset of the study and subsequentlytwice per week. The body weights of the mice are presented in Table 80and are expressed increase in grams over the PBS control weight takenbefore the start of treatment. Liver, spleen, and kidney weights weremeasured at the end of the study, and are also presented in Table 80 aspercentage of the body weight. Those antisense oligonucleotides whichdid not affect more than six-fold increases in liver and spleen weightabove the PBS control were selected for further studies.

TABLE 80 Change in body and organ weights of CD1 mice after antisenseoligonucleotide treatment body Liver Kidney Spleen weight (%) (%) (%)(g) PBS 5 1.5 0.3 7 ISIS 416850 6 1.6 0.4 12 ISIS 416858 7 1.6 0.6 12ISIS 416864 5 1.6 0.3 12 ISIS 412223 6 1.5 0.4 12 ISIS 412224 6 1.6 0.510 ISIS 412225 6 1.5 0.4 10 ISIS 413481 6 1.5 0.5 9 ISIS 413482 6 1.60.5 11 ISIS 416848 6 1.5 0.4 11 ISIS 416849 8 1.5 0.4 8 ISIS 416851 71.5 0.5 11 ISIS 416852 6 1.5 0.4 10 ISIS 416853 8 1.5 0.7 13 ISIS 4168547 1.2 0.4 13 ISIS 416855 8 1.4 0.6 12 ISIS 416856 6 1.4 1 0.4 10 ISIS416857 7 1.6 0.5 10 ISIS 416859 6 1.5 0.4 10 ISIS 416860 6 1.4 0.4 10ISIS 416861 5 1.3 0.4 9 ISIS 416862 6 1.5 0.4 10 ISIS 416863 5 1.5 0.4 9ISIS 416865 6 1.5 0.4 8 ISIS 416866 5 1.6 0.4 10 ISIS 416867 5 1.4 0.4 9

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Measurements of alanine transaminase (ALT) and aspartate transaminase(AST) are expressed in IU/L and the results are presented in Table 81.Those antisense oligonucleotides which did not affect an increase inALT/AST levels above seven-fold of control levels were selected forfurther studies. Plasma levels of bilirubin, cholesterol and albuminwere also measured using the same clinical chemistry analyzer and arepresented in Table 81 expressed in mg/dL. Those antisenseoligonucleotides which did not affect an increase in levels of bilirubinmore than two-fold of the control levels by antisense oligonucleotidetreatment were selected for further studies.

TABLE 81 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of CD1 mice ALT AST Bilirubin Albumin Cholesterol(IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 32 68 0.25 3.7 135 ISIS 41685075 99 0.21 3.5 142 ISIS 416858 640 547 0.28 4.4 181 ISIS 416864 36 670.19 2.6 152 ISIS 412223 60 125 0.20 3.0 117 ISIS 412224 214 183 0.193.4 114 ISIS 412225 40 69 0.23 3.3 128 ISIS 413481 85 143 0.18 3.2 153ISIS 413482 54 77 0.24 3.0 138 ISIS 416848 153 153 0.19 3.1 151 ISIS416849 1056 582 0.22 2.5 109 ISIS 416851 47 76 0.19 3.1 106 ISIS 41685249 91 0.16 4.9 125 ISIS 416853 1023 1087 0.25 3.1 164 ISIS 416854 16131140 0.21 5.5 199 ISIS 416855 786 580 0.25 4.2 162 ISIS 416856 130 1290.23 5.2 109 ISIS 416857 370 269 0.22 3.7 94 ISIS 416859 214 293 0.204.2 160 ISIS 416860 189 160 0.23 3.5 152 ISIS 416861 38 85 0.27 4.3 133ISIS 416862 225 172 0.36 3.9 103 ISIS 416863 41 101 0.24 3.6 118 ISIS416865 383 262 0.27 4.1 95 ISIS 416866 36 120 0.29 4.3 113 ISIS 41686745 82 0.21 3.3 144

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) were measured usingan automated clinical chemistry analyzer and results are presented inTable 82 expressed in mg/dL. Those antisense oligonucleotides which didnot affect more than a two-fold increase in BUN levels compared to thePBS control were selected for further studies.

TABLE 82 Effect of antisense oligonucleotide treatment on BUN levels(mg/dL) in the kidney of CD1 mice BUN PBS 22 ISIS 416850 24 ISIS 41685823 ISIS 416864 24 ISIS 412223 28 ISIS 412224 29 ISIS 412225 23 ISIS413481 23 ISIS 413482 27 ISIS 416848 23 ISIS 416849 23 ISIS 416851 21ISIS 416852 21 ISIS 416853 22 ISIS 416854 27 ISIS 416855 23 ISIS 41685621 ISIS 416857 17 ISIS 416859 18 ISIS 416860 25 ISIS 416861 23 ISIS416862 21 ISIS 416863 22 ISIS 416865 20 ISIS 416866 22 ISIS 416867 20

Hematology Assays

Blood obtained from all the mice groups were sent to Antech Diagnosticsfor hematocrit (HCT) measurements, as well as measurements of variousblood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC,and platelets, as well as total hemoglobin content analysis. The resultsare presented in Tables 83 and 84. Those antisense oligonucleotideswhich did not affect a decrease in platelet count of more than 50% andan increase in monocyte count of more than three-fold were selected forfurther studies.

TABLE 83 Effect of antisense oligonucleotide treatment on hematologicfactors in CD1 mice RBC Hemoglobin HCT WBC (10⁶/μL) (g/dL) (%) (10³/μL)PBS 10 15 51 7 ISIS 416850 10 15 49 5 ISIS 416858 9 14 50 8 ISIS 41686410 15 52 5 ISIS 412223 9 15 48 7 ISIS 412224 10 15 50 9 ISIS 412225 9 1550 7 ISIS 413481 9 13 45 7 ISIS 413482 10 15 50 8 ISIS 416848 9 14 47 7ISIS 416849 9 14 48 9 ISIS 416851 9 14 47 6 ISIS 416852 9 14 49 5 ISIS416853 11 17 56 8 ISIS 416854 9 13 43 12 ISIS 416855 9 14 50 6 ISIS416856 9 14 47 5 ISIS 416857 10 15 53 6 ISIS 416859 10 15 49 6 ISIS416860 10 15 51 7 ISIS 416861 9 14 48 7 ISIS 416862 9 14 49 6 ISIS416863 9 14 48 7 ISIS 416865 9 14 50 7 ISIS 416866 9 15 51 6 ISIS 41686710 14 47 8

TABLE 84 Effect of antisense oligonucleotide treatment on blood cellcount in CD1 mice Neutrophil Lymphocyte Monocytes Platelets (cells/μL)(cells/μL) (cells/μL) (10³/μL) PBS 1023 6082 205 940 ISIS 416850 11444004 156 916 ISIS 416858 2229 5480 248 782 ISIS 416864 973 3921 141 750ISIS 412223 1756 4599 200 862 ISIS 412224 2107 6284 195 647 ISIS 4122251547 4969 293 574 ISIS 413481 1904 4329 204 841 ISIS 413482 1958 5584275 818 ISIS 416848 1264 5268 180 953 ISIS 416849 1522 6967 253 744 ISIS416851 1619 4162 194 984 ISIS 416852 1241 3646 189 903 ISIS 416853 20405184 225 801 ISIS 416854 2082 9375 455 1060 ISIS 416855 1443 4236 263784 ISIS 416856 1292 3622 151 753 ISIS 416857 1334 3697 215 603 ISIS416859 1561 4363 229 826 ISIS 416860 1291 4889 161 937 ISIS 416861 11225119 219 836 ISIS 416862 1118 4445 174 1007 ISIS 416863 1330 5617 2261131 ISIS 416865 1227 5148 315 872 ISIS 416866 1201 4621 211 1045 ISIS416867 1404 6078 188 1006

Example 38: Measurement of Half-Life of Antisense Oligonucleotide in CD1Mouse Liver

Fifteen antisense oligonucleotides which had been evaluated in CD1 mice(Example 37) were further evaluated. CD1 mice were treated with ISISantisense oligonucleotides and the oligonucleotide half-life as well theelapsed time for oligonucleotide degradation and elimination in theliver was evaluated.

Treatment

Groups of fifteen CD1 mice each were injected subcutaneously twice perweek for 2 weeks with 50 mg/kg of ISIS 412223, ISIS 412225, ISIS 413481,ISIS 413482, ISIS 416851, ISIS 416852, ISIS 416856, ISIS 416860, ISIS416861, ISIS 416863, ISIS 416866, ISIS 416867, ISIS 412224, ISIS 416848or ISIS 416859. Five mice from each group were sacrificed 3 days, 28days, and 56 days after the last dose, livers were collected foranalysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide was measured. Themethod used is a modification of previously published methods (Leeds etal., 1996; Geary et al., 1999) which consist of a phenol-chloroform(liquid-liquid) extraction followed by a solid phase extraction. Aninternal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modifiedphosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT,designated herein as SEQ ID NO: 270) was added prior to extraction.Tissue sample concentrations were calculated using calibration curves,with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g.The results are presented in Table 85 expressed as μg/g liver tissue.The half-life of each oligonucleotide was also presented in Table 85.

TABLE 85 Full-length oligonucleotide concentration and half-life in theliver of CD1 mice ISIS day day day Half-Life No Motif 3 28 56 (days)412223 5-10-5 276 127 52 21.9 412224 5-10-5 287 111 31 16.6 4122255-10-5 279 91 47 20.7 413481 5-10-5 185 94 31 20.6 413482 5-10-5 262 9540 19.5 416848 5-10-5 326 147 68 23.5 416851 5-10-5 319 147 68 23.8416852 5-10-5 306 145 83 28.4 416856 5-10-5 313 115 46 19.2 4168595-10-5 380 156 55 19.0 416860 5-10-5 216 96 36 20.6 416861 5-10-5 175 5939 24.5 416863 5-10-5 311 101 48 19.8 416866 5-10-5 246 87 25 16.0416867 5-10-5 246 87 35 18.9

Example 39: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in Sprague-Dawley Rats

Fifteen antisense oligonucleotides which had been evaluated in CD1 mice(Example 37) were further evaluated in Sprague-Dawley rats for changesin the levels of various metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice per week for 6 weeks with 50 mg/kg of ISIS 412223, ISIS 412224,ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416851, ISIS416852, ISIS 416856, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416863,ISIS 416866, or ISIS 416867. A control group of four Sprague Dawley ratswas injected subcutaneously with PBS twice per week for 6 weeks. Bodyweight measurements were taken before and throughout the treatmentperiod. Three days after the last dose, urine samples were collected andthe rats were then sacrificed, organ weights were measured, and bloodwas collected for further analysis.

Body Weight and Organ Weights

The body weights of the rats were measured at the onset of the study andsubsequently twice per week. The body weights are presented in Table 86and are expressed as increase in grams over the PBS control weight takenbefore the start of treatment. Liver, spleen and kidney weights weremeasured at the end of the study, and are also presented in Table 86 asa percentage of the body weight. Those antisense oligonucleotides whichdid not affect more than six-fold increases in liver and spleen weightabove the PBS control were selected for further studies.

TABLE 86 Change in body and organ weights of Sprague Dawley rats afterantisense oligonucleotide treatment Body weight Liver Kidney Spleen (g)(%) (%) (%) PBS 179 4 0.9 0.2 ISIS 412223 126 5 1.0 0.5 ISIS 412224 1655 1.0 0.5 ISIS 412225 184 4 1.0 0.5 ISIS 413481 147 5 0.9 0.3 ISIS413482 158 5 1.0 0.6 ISIS 416848 117 5 1.1 0.8 ISIS 416851 169 5 0.9 0.3ISIS 416852 152 5 1.0 0.4 ISIS 416856 156 5 1.0 0.4 ISIS 416859 128 41.0 0.4 ISIS 416860 123 5 1.0 0.5 ISIS 416861 182 5 0.9 0.3 ISIS 416863197 5 1.0 0.4 ISIS 416866 171 5 1.0 0.5 ISIS 416867 129 5 1.0 0.5

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Measurements of alanine transaminase (ALT) and aspartate transaminase(AST) are expressed in IU/L and the results are presented in Table 87.Those antisense oligonucleotides which did not affect an increase inALT/AST levels above seven-fold of control levels were selected forfurther studies. Plasma levels of bilirubin and albumin were alsomeasured using the same clinical chemistry analyzer and results arepresented in Table 87 and expressed in mg/dL. Those antisenseoligonucleotides which did not affect an increase in levels of bilirubinmore than two-fold of the control levels by antisense oligonucleotidetreatment were selected for further studies.

TABLE 87 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin(IU/L) (IU/L) (mg/dL) (mg/dL) PBS 42 71 0.13 4 ISIS 412223 85 180 0.14 5ISIS 412224 84 132 0.12 4 ISIS 412225 48 108 0.15 5 ISIS 413481 54 800.22 4 ISIS 413482 59 157 0.14 4 ISIS 416848 89 236 0.14 3 ISIS 41685164 91 0.14 4 ISIS 416852 49 87 0.15 4 ISIS 416856 123 222 0.13 4 ISIS416859 114 206 0.21 5 ISIS 416860 70 157 0.15 4 ISIS 416861 89 154 0.155 ISIS 416863 47 78 0.13 4 ISIS 416866 41 78 0.16 4 ISIS 416867 47 1260.17 4

Kidney Function

To evaluate the effect of ISIS oligonucleotides on the kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.). Results are presented in Table 88, expressed inmg/dL. Those antisense oligonucleotides which did not affect more than atwo-fold increase in BUN levels compared to the PBS control wereselected for further studies. The total urine protein and ratio of urineprotein to creatinine in total urine samples after antisenseoligonucleotide treatment was calculated and is also presented in Table88. Those antisense oligonucleotides which did not affect more than afive-fold increase in urine protein/creatinine ratios compared to thePBS control were selected for further studies.

TABLE 88 Effect of antisense oligonucleotide treatment on metabolicmarkers in the kidney of Sprague-Dawley rats Total Urine urine protein/BUN Creatinine protein creatinine (mg/dL) (mg/dL) (mg/dL) ratio PBS 1938 60 1.7 ISIS 412223 24 46 224 4.6 ISIS 412224 24 44 171 3.8 ISIS412225 23 58 209 4.0 ISIS 413481 26 45 148 3.6 ISIS 413482 23 34 157 4.8ISIS 416848 26 64 231 3.9 ISIS 416851 24 70 286 4.0 ISIS 416852 25 60189 3.0 ISIS 416856 23 48 128 2.7 ISIS 416859 24 44 144 3.3 ISIS 41686023 58 242 4.6 ISIS 416861 22 39 205 5.1 ISIS 416863 29 73 269 3.8 ISIS416866 22 85 486 6.2 ISIS 416867 22 70 217 3.1

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics forhematocrit (HCT) measurements, as well as measurements of the variousblood cells, such as WBC (neutrophils and lymphocytes), RBC, andplatelets, and total hemoglobin content. The results are presented inTables 89 and 90. Those antisense oligonucleotides which did not affecta decrease in platelet count of more than 50% and an increase inmonocyte count of more than three-fold were selected for furtherstudies.

TABLE 89 Effect of antisense oligonucleotide treatment on hematologicfactors in Sprague-Dawley rats RBC Hemoglobin HCT WBC (10⁶/μL) (g/dL)(%) (10³/μL) PBS 6.9 13.2 42 9 ISIS 412223 7.2 13.1 41 20 ISIS 4122247.4 13.4 42 20 ISIS 412225 7.4 13.4 42 15 ISIS 413481 7.5 14.2 43 14ISIS 413482 7.1 13.2 40 13 ISIS 416848 6.0 11.1 35 17 ISIS 416851 7.413.7 42 11 ISIS 416852 7.2 13.4 42 13 ISIS 416856 7.7 14.1 43 19 ISIS416859 7.8 14.0 45 16 ISIS 416860 7.8 14.1 45 17 ISIS 416861 7.7 14.6 4515 ISIS 416863 7.6 14.1 45 17 ISIS 416866 7.8 14.0 44 20 ISIS 416867 7.814.0 45 14

TABLE 90 Effect of antisense oligonucleotide treatment on blood cellcount in Sprague-Dawley rats Neutrophil Lymphocyte Platelets (/mL) (/mL)(10³/mL) PBS 988 7307 485 ISIS 412223 1826 16990 567 ISIS 412224 186516807 685 ISIS 412225 1499 13204 673 ISIS 413481 1046 12707 552 ISIS413482 1125 11430 641 ISIS 416848 1874 14316 384 ISIS 416851 1001 9911734 ISIS 416852 836 11956 632 ISIS 416856 3280 14328 740 ISIS 4168591414 14323 853 ISIS 416860 1841 13986 669 ISIS 416861 1813 12865 1008ISIS 416863 1720 14669 674 ISIS 416866 1916 16834 900 ISIS 416867 304410405 705

Example 40: Measurement of Half-Life of Antisense Oligonucleotide in theLiver and Kidney of Sprague-Dawley Rats

Sprague Dawley rats were treated with ISIS antisense oligonucleotidestargeting human Factor 11 and the oligonucleotide half-life as well asthe elapsed time for oligonucleotide degradation and elimination fromthe liver and kidney was evaluated.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice per week for 2 weeks with 20 mg/kg of ISIS 412223, ISIS 412224,ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416851, ISIS416852, ISIS 416856, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416863,ISIS 416866, or ISIS 416867. Three days after the last dose, the ratswere sacrificed, and livers and kidneys were harvested.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. The results are presented in Tables 91 and 92,expressed as μg/g liver or kidney tissue. Half-lives were thencalculated using WinNonlin software (PHARSIGHT).

TABLE 91 Full-length oligonucleotide concentration (μg/g) in the liverand kidney of Sprague-Dawley rats ISIS No Motif Kidney Liver 4122235-10-5 551 97 412224 5-10-5 487 107 412225 5-10-5 202 119 413481 5-10-5594 135 413482 5-10-5 241 95 416848 5-10-5 488 130 416851 5-10-5 264 193416852 5-10-5 399 108 416856 5-10-5 378 84 416859 5-10-5 253 117 4168605-10-5 247 94 416861 5-10-5 187 159 416863 5-10-5 239 82 416866 5-10-5210 98 416867 5-10-5 201 112

TABLE 92 Total oligonucleotide concentration (μg/g) in the liver andkidney of Sprague-Dawley rats ISIS No Motif Kidney Liver 412223 5-10-5395 86 412224 5-10-5 292 78 412225 5-10-5 189 117 413481 5-10-5 366 96413482 5-10-5 217 91 416848 5-10-5 414 115 416851 5-10-5 204 178 4168525-10-5 304 87 416856 5-10-5 313 80 416859 5-10-5 209 112 416860 5-10-5151 76 416861 5-10-5 165 144 416863 5-10-5 203 79 416866 5-10-5 145 85416867 5-10-5 157 98

TABLE 93 Half-life (days) of ISIS oligonucleotides in the liver andkidney of Sprague-Dawley rats ISIS No Motif Half-life 412223 5-10-5 22412224 5-10-5 17 412225 5-10-5 21 413481 5-10-5 21 413482 5-10-5 20416848 5-10-5 24 416851 5-10-5 24 416852 5-10-5 28 416856 5-10-5 19416859 5-10-5 19 416860 5-10-5 21 416861 5-10-5 25 416863 5-10-5 20416866 5-10-5 16 416867 5-10-5 19

Example 41: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in CD1 Mice

ISIS oligonucleotides with 6-8-6 MOE and 5-8-5 MOE motifs targetinghuman Factor 11 were administered in CD1 mice evaluated for changes inthe levels of various metabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice per weekfor 6 weeks with 50 mg/kg of ISIS 416850, ISIS 445498, ISIS 445503, ISIS445504, ISIS 445505, ISIS 445509, ISIS 445513, ISIS 445522, ISIS 445530,ISIS 445531, or ISIS 445532. A control group of five CD1 mice wasinjected subcutaneously with PBS twice per week for 6 weeks. Body weightmeasurements were taken before and at the end of the treatment period.Three days after the last dose, the mice were sacrificed, organ weightswere measured, and blood was collected for further analysis.

Body Weight and Organ Weight

The body weight changes in the mice are presented in Table 94 and areexpressed increase in grams over the PBS control weight taken before thestart of treatment. Liver, spleen and kidney weights were measured atthe end of the study, and are also presented in Table 94 as percentageof the body weight. Those antisense oligonucleotides which did notaffect more than six-fold increases in liver and spleen weight above thePBS control were selected for further studies.

TABLE 94 Change in body and organ weights of CD1 mice after antisenseoligonucleotide treatment Body weight Liver Kidney Spleen (g) (%) (%)(%) PBS 10 5 1.6 0.3 ISIS 416850 11 6 1.5 0.4 ISIS 445498 10 6 1.6 0.5ISIS 445503 9 8 1.4 0.6 ISIS 445504 11 6 1.6 0.4 ISIS 445505 12 6 1.50.5 ISIS 445509 10 6 1.6 0.5 ISIS 445513 9 5 1.6 0.4 ISIS 445522 11 61.7 0.4 ISIS 445530 11 6 1.5 0.5 ISIS 445531 10 6 1.5 0.5 ISIS 445532 106 1.6 0.4

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Measurements of alanine transaminase (ALT) and aspartate transaminase(AST) are expressed in IU/L and the results are presented in Table 95.Those antisense oligonucleotides which did not affect an increase inALT/AST levels above seven-fold of control levels were selected forfurther studies. Plasma levels of bilirubin and albumin were alsomeasured and results are also presented in Table 95 and expressed inmg/dL. Those antisense oligonucleotides which did not affect an increasein levels of bilirubin more than two-fold of the control levels byantisense oligonucleotide treatment were selected for further studies.

TABLE 95 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of CD1 mice ALT AST Bilirubin Albumin (IU/L) (IU/L)(mg/dL) (mg/dL) PBS 34 49 0.23 3.6 ISIS 416850 90 115 0.20 3.2 ISIS445498 66 102 0.24 3.4 ISIS 445503 1314 852 0.28 3.4 ISIS 445504 71 1070.17 3.4 ISIS 445505 116 153 0.18 3.2 ISIS 445509 80 117 0.17 3.1 ISIS445513 37 84 0.22 3.1 ISIS 445522 51 110 0.19 3.4 ISIS 445530 104 1360.18 3.2 ISIS 445531 60 127 0.16 3.2 ISIS 445532 395 360 0.20 2.9

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) were measured usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). Results are presented in Table 96, expressed in mg/dL.Those antisense oligonucleotides which did not affect more than atwo-fold increase in BUN levels compared to the PBS control wereselected for further studies.

TABLE 96 Effect of antisense oligonucleotide treatment on BUN levels(mg/dL) in the kidney of CD1 mice BUN PBS 29 ISIS 416850 28 ISIS 44549828 ISIS 445503 29 ISIS 445504 29 ISIS 445505 29 ISIS 445509 29 ISIS445513 27 ISIS 445522 28 ISIS 445530 26 ISIS 445531 27 ISIS 445532 23

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics forhematocrit (HCT) measurements, as well as measurements of the variousblood cells, such as WBC (neutrophils and lymphocytes), RBC, andplatelets, and total hemoglobin content. The results are presented inTables 97 and 98. Those antisense oligonucleotides which did not affecta decrease in platelet count of more than 50% and an increase inmonocyte count of more than three-fold were selected for furtherstudies.

TABLE 97 Effect of antisense oligonucleotide treatment on hematologicfactors in CD1 mice RBC Hemoglobin HCT WBC (10⁶/mL) (g/dL) (%) (10³/mL)PBS 9.6 15.0 51 6 ISIS 416850 9.8 14.8 50 6 ISIS 445498 9.4 13.9 47 5ISIS 445503 9.2 13.6 46 8 ISIS 445504 9.6 14.7 49 5 ISIS 445505 9.6 14.649 5 ISIS 445509 10.2 15.3 51 5 ISIS 445513, 9.8 15.0 50 7 ISIS 4455229.7 14.6 49 5 ISIS 445530 10.0 15.1 50 7 ISIS 445531 9.4 14.5 48 9 ISIS445532 9.7 14.8 48 7

TABLE 98 Effect of antisense oligonucleotide treatment on blood cellcount in CD1 mice Neutrophil Lymphocyte Platelets (/mL) (/mL) (10³/mL)PBS 1356 4166 749 ISIS 416850 1314 4710 614 ISIS 445498 1197 3241 802ISIS 445503 1475 6436 309 ISIS 445504 959 3578 826 ISIS 445505 818 3447725 ISIS 445509 1104 3758 1085 ISIS 445513 959 5523 942 ISIS 445522 6983997 1005 ISIS 445530 930 5488 849 ISIS 445531 2341 6125 996 ISIS 4455321116 5490 689

Example 42: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in Sprague-Dawley Rats

Eight antisense oligonucleotides which had been evaluated in CD1 mice(Example 41) were further evaluated in Sprague-Dawley rats for changesin the levels of various metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice per week for 6 weeks with 50 mg/kg of ISIS 445498, ISIS 445504,ISIS 445505, ISIS 445509, ISIS 445513, ISIS 445522, ISIS 445530, or ISIS445531. A control group of Sprague Dawley rats was injectedsubcutaneously with PBS twice per week for 6 weeks. Body weightmeasurements were taken before and throughout the treatment period.Three days after the last dose, urine samples were collected and therats were then sacrificed, organ weights were measured, and blood wascollected for further analysis.

Body Weight and Organ Weight

The body weights of the rats were measured at the onset of the study andsubsequently twice per week. The body weights are presented in Table 99and are expressed as percent increase over the PBS control weight takenbefore the start of treatment. Liver, spleen and kidney weights weremeasured at the end of the study, and are also presented in Table 99 asa percentage of the body weight. Those antisense oligonucleotides whichdid not affect more than six-fold increases in liver and spleen weightabove the PBS control were selected for further studies.

TABLE 99 Change in body and organ weights of Sprague Dawley rats afterantisense oligonucleotide treatment (%) Body weight Liver Spleen KidneyISIS 445498 −17 +26 +107 −10 ISIS 445504 −15 +22 +116  +6 ISIS 445505−21 +12 +146  +2 ISIS 445509 −17 +16 +252  +3 ISIS 445513 −13 +25 +194+15 ISIS 445522 −13 +26 +184 +19 ISIS 445530  −7 +24  +99  +4 ISIS445531 −10 +17  +89  +4

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 100expressed in IU/L. Those antisense oligonucleotides which did not affectan increase in ALT/AST levels above seven-fold of control levels wereselected for further studies. Plasma levels of bilirubin and albuminwere also measured using the same clinical chemistry analyzer; resultsare presented in Table 100 and expressed in mg/dL. Those antisenseoligonucleotides which did not affect an increase in levels of bilirubinmore than two-fold of the control levels by antisense oligonucleotidetreatment were selected for further studies.

TABLE 100 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin(IU/L) (IU/L) (mg/dL) (mg/dL) PBS 102 36 0.13 3.7 ISIS 445498 417 1240.14 3.7 ISIS 445504 206 86 0.11 3.5 ISIS 445505 356 243 0.15 3.6 ISIS445509 676 291 0.14 3.5 ISIS 445513 214 91 0.15 3.5 ISIS 445522 240 1380.47 3.6 ISIS 445530 116 56 0.11 3.7 ISIS 445531 272 137 0.12 3.7

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.). Results are presented in Table 101, expressedin mg/dL. Those antisense oligonucleotides which did not affect morethan a two-fold increase in BUN levels compared to the PBS control wereselected for further studies. The total urine protein and ratio of urineprotein to creatinine in total urine samples after antisenseoligonucleotide treatment was calculated and is also presented in Table101. Those antisense oligonucleotides which did not affect more than afive-fold increase in urine protein/creatinine ratios compared to thePBS control were selected for further studies.

TABLE 101 Effect of antisense oligonucleotide treatment on metabolicmarkers in the kidney of Sprague-Dawley rats Urine BUN Creatinineprotein/creatinine (mg/dL) (mg/dL) ratio PBS 18 0.4 1.4 ISIS 445498 250.5 3.1 ISIS 445504 26 0.4 4.3 ISIS 445505 24 0.4 3.8 ISIS 445509 27 0.54.0 ISIS 445513 24 0.4 4.6 ISIS 445522 25 0.4 6.4 ISIS 445530 22 0.4 4.2ISIS 445531 23 0.4 3.4

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics forhematocrit (HCT) measurements, as well as measurements of the variousblood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC,and platelets, and total hemoglobin content. The results are presentedin Tables 102 and 103. Those antisense oligonucleotides which did notaffect a decrease in platelet count of more than 50% and an increase inmonocyte count of more than three-fold were selected for furtherstudies.

TABLE 102 Effect of antisense oligonucleotide treatment on hematologicfactors in Sprague-Dawley rats RBC Hemoglobin HCT WBC (/pL) (g/dL) (%)(/nL) PBS 8.8 16.0 55 13 ISIS 445498 8.5 14.7 49 13 ISIS 445504 8.9 14.750 16 ISIS 445505 9.1 15.0 50 21 ISIS 445509 8.4 14.1 47 17 ISIS 4455137.8 13.0 44 17 ISIS 445522 7.7 13.6 47 18 ISIS 445530 8.9 14.7 50 12ISIS 445531 8.8 14.8 50 13

TABLE 103 Effect of antisense oligonucleotide treatment on blood cellcount in Sprague-Dawley rats Neutrophil Lymphocyte Monocytes Platelets(%) (%) (%) (/nL) PBS 14 82 2.0 1007 ISIS 445498  9 89 2.0 1061 ISIS445504 10 87 2.0  776 ISIS 445505 10 87 2.5 1089 ISIS 445509 11 84 3.81115 ISIS 445513 14 82 3.5 1051 ISIS 445522 13 84 2.8 1334 ISIS 44553011 87 2.0 1249 ISIS 445531 10 86 2.8 1023

Example 43: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in CD1 Mice

ISIS oligonucleotides with 4-8-4 MOE, 3-8-3 MOE, 2-10-2 MOE, 3-10-3 MOE,and 4-10-4 MOE motifs targeting human Factor 11 were administered in CD1mice evaluated for changes in the levels of various metabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice per weekfor 6 weeks with 50 mg/kg of ISIS 449707, ISIS 449708, ISIS 449409, ISIS449710, or ISIS 449711. A control group of five CD1 mice was injectedsubcutaneously with PBS twice per week for 6 weeks. Body weightmeasurements were taken before and at the end of the treatment period.Three days after the last dose, the mice were sacrificed, organ weightswere measured, and blood was collected for further analysis.

Body Weight and Organ Weight

The body weights of the mice taken at the end of the study are presentedin Table 104 and are expressed in grams. Liver, spleen and kidneyweights were also measured at the end of the study and are alsopresented in Table 104 as percentage of the body weight. Those antisenseoligonucleotides which did not affect more than six-fold increases inliver and spleen weight above the PBS control were selected for furtherstudies.

TABLE 104 Change in body and organ weights of CD1 mice after antisenseoligonucleotide treatment Body weight Liver Spleen Kidney (g) (%) (%)(%) PBS 39 — — — ISIS 449707 42 +11 +63 −5 ISIS 449708 40 +17 +66 0 ISIS449709 40 +15 +62 −14 ISIS 449710 42  +6 +43 −7 ISIS 449711 42 +18 +63−12

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 105expressed in IU/L. Those antisense oligonucleotides which did not affectan increase in ALT/AST levels above seven-fold of control levels wereselected for further studies. Plasma levels of bilirubin and albuminwere also measured using the same clinical chemistry analyzer andresults are presented in Table 105 and expressed in mg/dL. Thoseantisense oligonucleotides which did not affect an increase in levels ofbilirubin more than two-fold of the control levels by antisenseoligonucleotide treatment were selected for further studies.

TABLE 105 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of CD1 mice ALT AST Bilirubin Albumin (IU/L) (IU/L)(mg/dL) (mg/dL) PBS 39 52 0.22 3.2 ISIS 449707 41 62 0.19 2.3 ISIS449708 66 103  0.17 2.8 ISIS 449709 62 83 0.18 2.8 ISIS 449710 43 950.18 2.8 ISIS 449711 52 83 0.22 2.8

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.). Results are presented in Table 106, expressedin mg/dL. Those antisense oligonucleotides which did not affect morethan a two-fold increase in BUN levels compared to the PBS control wereselected for further studies.

TABLE 106 Effect of antisense oligonucleotide treatment on metabolicmarkers (mg/dL) in the kidney of CD1 mice BUN Creatinine PBS 28 0.3 ISIS449707 27 0.2 ISIS 449708 28 0.2 ISIS 449709 34 0.3 ISIS 449710 29 0.2ISIS 449711 26 0.2

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics forhematocrit (HCT), measurements, as well as measurements of the variousblood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC,and platelets, and total hemoglobin content. The results are presentedin Tables 107 and 108. Those antisense oligonucleotides which did notaffect a decrease in platelet count of more than 50% and an increase inmonocyte count of more than three-fold were selected for furtherstudies.

TABLE 107 Effect of antisense oligonucleotide treatment on hematologicfactors in CD1 mice RBC Hemoglobin Hematocrit WBC (/pL) (g/dL) (%) (/nL)PBS 9.8 14.6 54 6 ISIS 449707 8.4 12.4 45 6 ISIS 449708 9.2 13.2 48 7ISIS 449709 9.2 13.2 49 5 ISIS 449710 9.1 13.5 48 7 ISIS 449711 9.0 13.348 6

TABLE 108 Effect of antisense oligonucleotide treatment on blood cellcount in CD1 mice Neutrophils Lymphocytes Monocytes Platelets (%) (%)(%) / (/nL) PBS 15 80 3 1383 ISIS 449707 11 85 3 1386 ISIS 449708 17 775 1395 ISIS 449709 19 76 4 1447 ISIS 449710 15 81 3 1245 ISIS 449711 1579 6 1225

Example 44: Tolerability of Antisense Oligonucleotides Targeting HumanFactor 11 in Sprague-Dawley Rats

Five antisense oligonucleotides which had been evaluated in CD1 mice(Example 43) were further evaluated in Sprague-Dawley rats for changesin the levels of various metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice per week for 6 weeks with 50 mg/kg of ISIS 449707, ISIS 449708,ISIS 449709, ISIS 449710, or ISIS 449711. A control group of fourSprague Dawley rats was injected subcutaneously with PBS twice per weekfor 6 weeks. Body weight measurements were taken before and throughoutthe treatment period. Three days after the last dose, urine samples werecollected and the rats were then sacrificed, organ weights weremeasured, and blood was collected for further analysis.

Body Weight and Organ Weight

The body weights of the rats were measured at the onset of the study andat the end of the study. The body weight changes are presented in Table109 and are expressed as increase in grams over the PBS control weighttaken before the start of treatment. Liver, spleen and kidney weightswere measured at the end of the study, and are also presented in Table109 as a percentage of the body weight. Those antisense oligonucleotideswhich did not affect more than six-fold increases in liver and spleenweight above the PBS control were selected for further studies.

TABLE 109 Change in body and organ weights of Sprague Dawley rats afterantisense oligonucleotide treatment Body weight Liver Spleen Kidney (g)(%) (%) (%) PBS 478 — — — ISIS 449707 352 +41 +400 +80 ISIS 449708 382+31 +259 +40 ISIS 449709 376  +8 +231 +19 ISIS 449710 344 +82 +302 +50ISIS 449711 362 +52 +327 +72

Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function,plasma concentrations of ALT and AST were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of alanine transaminase (ALT) and aspartatetransaminase (AST) were measured and the results are presented in Table110 expressed in IU/L. Those antisense oligonucleotides which did notaffect an increase in ALT/AST levels above seven-fold of control levelswere selected for further studies. Plasma levels of bilirubin andalbumin were also measured and results are presented in Table 110 andexpressed in mg/dL. Those antisense oligonucleotides which did notaffect an increase in levels of bilirubin more than two-fold of thecontrol levels by antisense oligonucleotide treatment were selected forfurther studies.

TABLE 110 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin(IU/L) (IU/L) (mg/dL) (mg/dL) PBS 41 107 0.1 3.4 ISIS 449707 61 199 0.23.1 ISIS 449708 25  90 0.1 3.2 ISIS 449709 63 126 0.2 3.1 ISIS 449710 36211 0.1 2.9 ISIS 449711 32 163 0.1 2.9

Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function,plasma concentrations of BUN and creatinine were measured using anautomated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,N.Y.). Results are presented in Table 111, expressed in mg/dL. Thoseantisense oligonucleotides which did not affect more than a two-foldincrease in BUN levels compared to the PBS control were selected forfurther studies. The total urine protein and ratio of urine protein tocreatinine in total urine samples after antisense oligonucleotidetreatment was calculated and is also presented in Table 111. Thoseantisense oligonucleotides which did not affect more than a five-foldincrease in urine protein/creatinine ratios compared to the PBS controlwere selected for further studies.

TABLE 111 Effect of antisense oligonucleotide treatment on metabolicmarkers in the kidney of Sprague-Dawley rats Urine BUN Creatinineprotein/creatinine (mg/dL) (mg/dL) ratio PBS 22 0.4 1.5 ISIS 449707 240.4 3.2 ISIS 449708 24 0.4 5.7 ISIS 449709 24 0.4 3.4 ISIS 449710 29 0.35.9 ISIS 449711 28 0.4 7.3

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics forhematocrit (HCT) measurements, as well as measurements of the variousblood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC,and platelets, and total hemoglobin content. The results are presentedin Tables 112 and 113. Those antisense oligonucleotides which did notaffect a decrease in platelet count of more than 50% and an increase inmonocyte count of more than three-fold were selected for furtherstudies.

TABLE 112 Effect of antisense oligonucleotide treatment on hematologicfactors in Sprague-Dawley rats RBC Hemoglobin Hematocrit WBC (/pL)(g/dL) (%) (/nL) PBS 8.2 15.1 50 16 ISIS 449707 6.0 12.0 40 20 ISIS449708 6.6 12.2 40 22 ISIS 449709 6.9 12.6 41 14 ISIS 449710 6.3 12.5 4113 ISIS 449711 6.4 12.6 43 13

TABLE 113 Effect of antisense oligonucleotide treatment on blood cellcount in Sprague-Dawley rats Neutrophils Lymphocytes Monocytes Platelets(%) (%) (%) (/nL) PBS 12 84 2 1004 ISIS 449707 6 91 2 722 ISIS 449708 692 2 925 ISIS 449709 5 91 3 631 ISIS 449710 6 91 2 509 ISIS 449711 7 902 919

Example 45: Dose-Dependent Pharmacologic Effect of AntisenseOligonucleotides Targeting Human Factor 11 in Cynomolgus Monkeys

Several antisense oligonucleotides were tested in cynomolgus monkeys todetermine the pharmacologic effects of the oligonucleotides on Factor 11activity, anticoagulation and bleeding times, liver and kidneydistributions, and tolerability. All the ISIS oligonucleotides used inthis study target human Factor 11 mRNA and are also fully cross-reactivewith the rhesus monkey gene sequence (see Table 44). It is expected thatthe rhesus monkey ISIS oligonucleotides are fully cross-reactive withthe cynomolgus monkey gene sequence as well. At the time the study wasundertaken, the cynomolgus monkey genomic sequence was not available inthe National Center for Biotechnology Information (NCBI) database;therefore, cross-reactivity with the cynomolgus monkey gene sequencecould not be confirmed.

Treatment

Groups, each consisting of two male and three female monkeys, wereinjected subcutaneously with ISIS 416838, ISIS 416850, ISIS 416858, ISIS416864, or ISIS 417002 in escalating doses. Antisense oligonucleotidewas administered to the monkeys at 5 mg/kg three times per a week forweek 1; 5 mg/kg twice per week for weeks 2 and 3; 10 mg/kg three timesper week for week 4; 10 mg/kg twice per week for weeks 5 and 6; 25 mg/kgthree times per week for week 7; and 25 mg/kg twice per week for weeks8, 9, 10, 11, and 12. One control group, consisting of two male andthree female monkeys, was injected subcutaneously with PBS according tothe same dosing regimen. An additional experimental group, consisting oftwo male and three female monkeys, was injected subcutaneously with ISIS416850 in a chronic, lower dose regimen. Antisense oligonucleotide wasadministered to the monkeys at 5 mg/kg three times per week for week 1;5 mg/kg twice per week for week 2 and 3; 10 mg/kg three times per weekfor week 4; and 10 mg/kg twice per week for weeks 5 to 12. Body weightswere measured weekly. Blood samples were collected 14 days and 5 daysbefore the start of treatment and subsequently once per week for Factor11 protein activity analysis in plasma, fibrinogen measurement, PT andaPTT measurements, bleeding times, and measurement of varioushematologic factors. On day 85, the monkeys were euthanized byexsanguination while under deep anesthesia, and organs harvested forfurther analysis.

RNA Analysis

On day 85, RNA was extracted from liver tissue for real-time PCRanalysis of Factor 11 using primer probe set LTS00301 (forward primersequence ACACGCATTAAAAAGAGCAAAGC, designated herein as SEQ ID NO 271;reverse primer sequence CAGTGTCATGGTAAAATGAAGAATGG, designated herein asSEQ ID NO: 272; and probe sequence TGCAGGCACAGCATCCCAGTGTTCTX,designated herein as SEQ ID NO. 273). Results are presented as percentinhibition of Factor 11, relative to PBS control. As shown in Table 114,treatment with ISIS oligonucleotides resulted in significant reductionof Factor 11 mRNA in comparison to the PBS control.

TABLE 114 Inhibition of Factor 11 mRNA in the cynomolgus monkey liverrelative to the PBS control % ISIS No inhibition 416838 37 416850 84416858 90 416864 44 417002 57

Protein Analysis

Plasma samples from all monkey groups taken on different days wereanalyzed by a sandwich-style ELISA assay (Affinity Biologicals Inc.)using an affinity-purified polyclonal anti-Factor 11 antibody as thecapture antibody and a peroxidase-conjugated polyclonal anti-Factor 11antibody as the detecting antibody. Monkey plasma was diluted 1:50 forthe assay. Peroxidase activity was expressed by incubation with thesubstrate o-phenylenediamine. The color produced was quantified using amicroplate reader at 490 nm and was considered to be proportional to theconcentration of Factor 11 in the samples.

The results are presented in Table 115, expressed as percentagereduction relative to that of the PBS control. Treatment with ISIS416850 and ISIS 416858 resulted in a time-dependent decrease in proteinlevels.

TABLE 115 Inhibition of Factor 11 protein in the cynomolgus monkey liverrelative to the PBS control ISIS ISIS ISIS ISIS ISIS ISIS Days 416838416850 416858 416864 417002 416850* −14 0 0 0 0 0 0 −5 0 0 0 5 0 1 8 3 86 7 0 6 15 4 4 16 9 4 13 22 5 11 23 7 2 12 29 8 15 28 10 8 20 36 11 1735 9 8 22 43 5 23 39 9 9 24 50 8 42 49 10 13 30 57 10 49 60 7 24 34 6411 55 68 5 26 37 71 12 57 71 10 30 41 78 10 63 73 9 22 42 85 10 64 78 823 34PT and aPTT Assay

Blood samples were collected in tubes containing sodium citrate. PT andaPTT were determined in duplicate with an ACL 9000 coagulationinstrument (Instrumentation Laboratory, Italy). The results wereinterpolated on a standard curve of serial dilutions citrated controlmonkey plasma tested to give a reported result in percent normal.

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT)were measured using platelet poor plasma (PPP) from monkeys treated withISIS oligonucleotides. PT and aPTT values are provided in Tables 116 and117 and are reported as International Normalized Ratio (INR) values. INRvalues for PT and aPTT were determined by dividing the PT or aPTT valuefor each experimental group by the PT or aPTT for the PBS treated group.This ratio was then raised to the power of the International SensitivityIndex (ISI) of the tissue factor used. The ISIS oligonucleotide, ISIS416850, given with the chronic dose regimen is distinguished from theother oligonucleotides with an asterisk (*).

As shown in Table 116, PT was not significantly prolonged in monkeystreated with ISIS oligonucleotides either in the escalating dose regimenor the chronic dose regimen. However, aPTT was prolonged in adose-dependent manner, as presented in Table 117. These data suggestthat antisense reduction of Factor 11 affects the contact activationpathway, but not the extrinsic pathway of blood coagulation. Therefore,antisense reduction of Factor 11 is useful for inhibiting the formationof a thrombus or clot in response to an abnormal vessel wall, but not inresponse to tissue injury.

TABLE 116 Effect of ISIS antisense oligonucleotides on PT ratio incynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS day 416838 416850416858 416864 417002 416850* −14 1.00 1.00 1.00 1.00 1.00 1.00 −5 1.001.00 1.00 1.00 1.00 1.00 8 1.03 1.00 1.05 1.02 1.02 1.03 15 1.03 1.021.07 1.07 1.04 1.06 22 1.07 1.02 1.06 1.03 1.04 1.06 29 1.03 1.03 1.081.06 1.01 1.00 36 1.05 1.02 1.07 1.06 1.05 1.06 43 1.03 1.01 1.08 1.041.03 1.02 50 1.02 1.02 1.03 1.01 0.99 0.98 57 1.04 1.04 1.09 1.08 1.03n.d. 64 1.04 1.03 1.09 1.10 1.03 n.d. 71 1.02 1.03 1.07 1.07 0.99 n.d.78 1.04 1.05 1.10 1.08 1.02 n.d. 85 1.05 1.04 1.07 1.13 1.02 n.d. n.d. =no data

TABLE 117 Effect of ISIS antisense oligonucleotides on aPTT ratio incynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS day 416838 416850416858 416864 417002 416850* −14 1.00 1.00 1.00 1.00 1.00 1.00 −5 1.001.00 1.00 1.00 1.00 1.00 8 1.07 1.05 1.03 1.05 1.05 1.12 15 1.05 1.051.07 1.03 1.03 1.07 22 1.20 1.13 1.18 1.11 1.16 1.21 29 1.19 1.13 1.201.13 1.11 1.26 36 1.20 1.26 1.36 1.19 1.18 1.34 43 1.18 1.17 1.28 1.071.06 1.22 50 1.25 1.68 1.55 1.26 1.18 1.35 57 1.21 1.59 1.59 1.19 1.22n.d. 64 1.18 1.64 1.60 1.12 1.11 n.d. 71 1.15 1.76 1.70 1.18 1.16 n.d.78 1.19 1.88 1.79 1.18 1.18 n.d. 85 1.22 1.99 1.76 1.25 1.20 n.d. n.d. =no data

Protein Activity Analysis

Blood samples were collected at various time points and Factor 11proenzyme was measured using a F11 assay based on clotting time.Clotting times were determined in duplicate with a ST4 semi-automatedcoagulation instrument (Diagnostica Stago, NJ). Thirty μl of citratedsample plasma diluted 1/20 in HEPES-NaCl buffer with BSA was incubatedwith 30 μl aPTT reagent (Automated aPTT, Organon Technika, NC) and 30 μlof citrated plasma deficient of Factor 11 (George King Bio-Medical Inc.)at 37° C. for 5 min, followed by the addition of 30 μl of 25 mM CaCl₂)to initiate clotting. Results were interpolated on a standard curve ofserially diluted citrated control plasma.

Results are presented in Table 118 as percent inhibition of Factor 11activity, relative to PBS control. The ISIS oligonucleotide, ISIS416850, given with the chronic dose regimen is distinguished from theother oligonucleotides with an asterisk (*).

TABLE 118 Inhibition of Factor 11 protein by ISIS antisenseoligonucleotides given in escalating dose/chronic dose regimen incynomolgus monkeys Days before/after ISIS ISIS ISIS ISIS ISIS ISIStreatment 416838 416850 416858 416864 417002 416850* −14 0 0 0 0 0 0 −50 0 0 5 0 1 8 3 8 6 7 0 6 15 4 4 16 9 4 13 22 5 11 23 7 2 12 29 8 15 2810 8 20 36 11 17 35 9 8 24 43 5 23 39 9 9 24 50 8 42 49 10 13 30 57 1049 60 7 24 n.d. 64 11 55 68 5 26 n.d. 71 12 57 71 10 30 n.d. 78 10 63 739 22 n.d. 85 10 64 78 8 23 n.d. n.d. = no data

Fibrinogen Assay

Nine parts of fresh monkey plasma was collected into one part oftrisodium citrate. The samples were evaluated of fibrinogen contentusing an ACL 9000 coagulation instrument (Instrumentation Laboratory,Italy). Results are presented in Table 119 expressed in mg/dL. The ISISoligonucleotide, ISIS 416850, given with the chronic dose regimen isdistinguished from the other oligonucleotides with an asterisk (*).

TABLE 119 Effect of ISIS antisense oligonucleotides on fibrinogen levelsin cynomolgus monkeys Days before/ after ISIS ISIS ISIS ISIS ISIS ISIStreatment PBS 416838 416850 416858 416864 417002 416850* −14 296 251 310277 300 291 274 −5 246 205 261 246 243 222 227 8 245 209 281 246 227 221232 15 207 198 270 219 210 195 174 22 219 183 243 222 184 199 192 29 231184 234 220 205 199 192 36 235 182 232 225 202 191 185 43 231 186 219229 198 187 194 50 251 216 215 259 233 236 204 57 235 190 186 225 200201 n.d. 64 240 190 190 236 218 236 n.d. 71 233 199 178 239 245 228 n.d.78 234 189 177 234 250 221 n.d. 85 246 196 187 243 240 224 n.d. n.d. =no data

Bleeding Assay

On different days during the treatment period, bleeding assay wasperformed using a Surgicutt Jr. device (ITC, New Jersey). Monkeys wereplaced in monkey chair with their arm placed in a steady support. Thearm was lightly shaved and a sphygmomanometer was placed on the upperarm. The cuff of the sphygmomanometer was inflated to 40 mm Hg and thispressure was maintained throughout the procedure. The area on the upperarm to be incised was cleansed with an antiseptic swab and the SurgicuttJr device was used to make an incision over the lateral aspect, volarsurface of the forearm, parallel to and 5 cm below the antecubitalcrease. At the exact moment the incision was made, a stopwatch wasstarted. Every 30 seconds, blood from the incision was blotted out usinga blotting paper without directly touching the incision, so thatformation of the platelet plug was not disturbed. Blood was blotted outevery 30 seconds until blood no longer stained the paper. The stopwatchwas then stopped and the bleeding time determined. The sphygmomanometerwas removed from the animal's arm, the incision site was antisepticallyswabbed and a wound closure strip applied. The results are provided inTable X, expressed in seconds. The results are provided in Table 120.The ISIS oligonucleotide, ISIS 416850, given with the chronic doseregimen is distinguished from the other oligonucleotides with anasterisk (*).

These data suggest that the hemorrhagic potential of the compoundsprovided herein is low.

TABLE 120 Bleeding assay in cynomolgus monkeys Days before/ after ISISISIS ISIS ISIS ISIS ISIS treatment PBS 416838 416850 416858 416864417002 416850* −14 147 200 172 154 166 185 177 −5 153 150 127 149 111175  93 15 111 167 165 146 153 174 149 22 113 165 151 100 133 194 143 36174 166 137 206 205 186 221 43 157 120 216 111 146 120 156 57 147 238195 138 216 206 n.d. 64 113 131 201 113 218 146 n.d. 78 114 145 203 186170 163 n.d. 85 147 201 201 191 203 182 n.d.

Platelet Aggregation Assay

Platelet aggregation was initiated by adding 1 mmol/L ADP and/or 3 μgcollagen (depending on the collection day, as outlined in Table 121) toplasma samples, and was allowed to proceed for 10 minutes. Aggregationwas characterized by recording the change in the electrical resistanceor impedance and the change in the initial slope of aggregation afterplatelet shape change. The aggregation test was performed twice persample on each collection day and the average value was taken. The ISISoligonucleotide, ISIS 416850, given with the chronic dose regimen isdistinguished from the other oligonucleotides with an asterisk (*).

TABLE 121 Effect of antisense oligonucleotide treatment on plateletaggregation in cynomolgus monkeys in Ohms day −5 day 15 day 36 day 43day 57 day 64 day 78 day 85 day 85 (with (with (with (with (with (with(with (with (with collagen) ADP) ADP) collagen) ADP) collagen) ADP) ADP)collagen) PBS 17 15 7 14 16 13 12 16 17 ISIS 15 15 8 16  7 13 11 15 24416838 ISIS 23 12 16 16 18 17  9 22 26 416850 ISIS 22 19 17 16 11 14  818 23 416858 ISIS 27 20 17.8 20 18 17 13 22 28 416864 ISIS 21 16 13.9 1918 18 18 22 24 417002 ISIS 21 14 11.6 21 n.d. n.d. n.d. n.d. n.d.416850* n.d. = no data

Body and Organ Weights

Body weights were taken once weekly throughout the dosing regimen. Themeasurements of each group are given in Table 122 expressed in grams.The results indicate that treatment with the antisense oligonucleotidesdid not cause any adverse changes in the health of the animals, whichmay have resulted in a significant alteration in weight compared to thePBS control. Organ weights were taken after the animals were euthanizedand livers, kidneys and spleens were harvested and weighed. The resultsare presented in Table 123 and also show no significant alteration inweights compared to the PBS control, except for ISIS 416858, which showsincrease in spleen weight. The ISIS oligonucleotide, ISIS 416850, givenwith the chronic dose regimen is distinguished from the otheroligonucleotides with an asterisk (*).

TABLE 122 Weekly measurements of body weights (g) of cynomolgus monkeysISIS ISIS ISIS ISIS ISIS ISIS day PBS 416838 416850 416858 416864 417002416850*  1 2780 2720 2572 2912 2890 2640 2665  8 2615 2592 2430 27402784 2523 2579 15 2678 2642 2474 2760 2817 2571 2607 22 2715 2702 25142800 2857 2617 2661 29 2717 2689 2515 2763 2863 2622 2667 36 2738 27082545 2584 3327 2631 2656 43 2742 2700 2544 2607 3355 2630 2670 50 27642731 2613 2646 3408 2652 2679 57 2763 2737 2629 2617 3387 2654 n.d. 642781 2746 2642 2618 3384 2598 n.d. 71 2945 2869 2769 2865 2942 2727 n.d.78 2815 2766 2660 2713 2822 2570 n.d. n.d. = no data

TABLE 123 Organ weights (g) of cynomolgus monkeys after antisenseoligonucleotide treatment Liver Spleen Kidney PBS 46 4 11 ISIS 416838 635 12 ISIS 416580 64 4 16 ISIS 416858 60 12 13 ISIS 416864 53 5 14 ISIS417002 51 5 15

Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Tables 124and 125 expressed in IU/L. Those antisense oligonucleotides which didnot affect an increase in ALT/AST levels above seven-fold of controllevels were selected for further studies. Plasma levels of bilirubinwere also measured and results are presented in Table 126 expressed inmg/dL. Those antisense oligonucleotides which did not affect an increasein levels of bilirubin more than two-fold of the control levels byantisense oligonucleotide treatment were selected for further studies.The ISIS oligonucleotide, ISIS 416850, given with the chronic doseregimen is distinguished from the other oligonucleotides with anasterisk (*).

TABLE 124 Effect of antisense oligonucleotide treatment on ALT (IU/L) inthe liver of cynomolgus monkeys Days before/ after ISIS ISIS ISIS ISISISIS ISIS treatment PBS 416838 416850 416858 416864 417002 416850* −1457 76 54 47 54 61 80 22 39 36 41 28 37 36 42 43 36 35 43 36 36 35 41 6438 40 60 47 43 42 n.d. 85 34 41 75 50 43 116  n.d. n.d. = no data

TABLE 125 Effect of antisense oligonucleotide treatment on AST (IU/L) inthe liver of cynomolgus monkeys Days before/ after ISIS ISIS ISIS ISISISIS ISIS treatment PBS 416838 416850 416858 416864 417002 416850* −1471 139  81 58 76 114  100 22 43 39 45 38 41 44  39 43 38 32 50 39 40 42 40 64 35 33 56 50 46 37 n.d. 85 41 30 82. 49 56 50 n.d. n.d. = no data

TABLE 126 Effect of antisense oligonucleotide treatment on bilirubin(mg/dL) in the liver of cynomolgus monkeys Days before/ after ISIS ISISISIS ISIS ISIS ISIS treatment PBS 416838 416850 416858 416864 417002416850* −14 0.24 0.26 0.21 0.27 0.31 0.26 0.28 22 0.16 0.17 0.13 0.180.22 0.20 0.19 43 0.17 0.17 0.13 0.14 0.17 0.21 0.18 64 0.19 0.15 0.140.12 0.16 0.14 n.d 85 0.20 0.13 0.14 0.14 0.17 0.12 n.d. n.d. = no data

Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function,urine samples were collected. The ratio of urine protein to creatininein urine samples after antisense oligonucleotide treatment wascalculated and is presented in Table 127. Those antisenseoligonucleotides which did not affect more than a five-fold increase inurine protein/creatinine ratios compared to the PBS control wereselected for further studies.

TABLE 127 Effect of antisense oligonucleotide treatment on urine proteinto creatinine ratio in cynomolgus monkeys Day 80 Day 84 PBS 0.09 0.10ISIS 416838 0.13 0.13 ISIS 416850 0.09 0.12 ISIS 416858 0.10 0.07 ISIS416864 0.36 0.34 ISIS 417002 0.18 0.24

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as theelapsed time oligonucleotide degradation and elimination from the liverand kidney were evaluated. The method used is a modification ofpreviously published methods (Leeds et al., 1996; Geary et al., 1999)which consist of a phenol-chloroform (liquid-liquid) extraction followedby a solid phase extraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. Half-lives were then calculated using WinNonlinsoftware (PHARSIGHT). The results are presented in Tables 128 and 129,expressed as μg/g liver or kidney tissue.

TABLE 128 Full-length oligonucleotide concentration (μg/g) in the liverand kidney of cynomolgus monkeys ISIS No. Kidney Liver 416838 1339 1087416850 2845 1225 416858 1772 1061 416864 2093 1275 417002 2162 1248

TABLE 129 Total oligonucleotide concentration (μg/g) in the liver andkidney of cynomolgus monkeys ISIS No. Kidney Liver 416838 1980 1544416850 3988 1558 416858 2483 1504 416864 3522 1967 417002 3462 1757

Hematology Assays

Blood obtained from all monkey groups were sent to Korea Institute ofToxicology (KIT) for HCT, MCV, MCH, and MCHC analysis, as well asmeasurements of the various blood cells, such as WBC (neutrophils,lymphocytes, monocytes, eosinophils, basophils, reticulocytes), RBC,platelets and total hemoglobin content. The results are presented inTables 130-143. Those antisense oligonucleotides which did not affect adecrease in platelet count of more than 50% and an increase in monocytecount of more than three-fold were selected for further studies. TheISIS oligonucleotide, ISIS 416850, given with the chronic dose regimenis distinguished from the other oligonucleotides with an asterisk (*).

TABLE 130 Effect of antisense oligonucleotide treatment on WBC count(×10³/μL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 14 12 13 14 13 13 15 day −513 12 13 14 13 14 15 day 8 10 10 10 12 11 10 13 day 15 10 10 9 11 10 1016 day 22 12 11 10 11 10 10 15 day 29 11 11 11 12 10 10 14 day 36 10 1010 12 10 11 16 day 43 10 10 9 11 10 10 15 day 50 12 11 11 13 12 13 15day 57 11 12 11 13 12 12 n.d. day 64 11 13 11 12 11 11 n.d. day 71 15 1515 13 14 12 n.d. day 78 10 11 12 11 11 9 n.d. day 85 10 12 15 11 12 10n.d. n.d. = no data

TABLE 131 Effect of antisense oligonucleotide treatment on RBC count(×10⁶/μL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 5.7 5.6 5.3 5.6 5.5 5.6 5.5day −5 5.7 5.6 5.5 5.6 5.6 5.6 5.5 day 8 5.7 5.7 5.4 5.6 5.7 5.6 5.5 day15 5.6 5.6 5.3 5.4 5.7 5.4 5.3 day 22 5.5 5.4 5 5.3 5.3 5.2 5.1 day 295.6 5.3 4.9 5.3 5.3 5.2 5.2 day 36 5.7 5.5 5.3 5.5 5.6 5.4 5.3 day 435.7 5.6 5.2 5.5 5.5 5.4 5.2 day 50 5.8 5.5 5.2 5.5 5.6 5.4 5.3 day 575.7 5.5 5.2 5.6 5.5 4.9 n.d. day 64 5.8 5.6 5.4 5.7 5.6 5.4 n.d day 715.6 5.5 5.4 5.6 5.6 5.5 n.d. day 78 5.6 5.4 5.3 5.4 5.3 5.4 n.d. day 855.6 5.5 5.5 5.5 5.4 5.4 n.d. n.d. = no data

TABLE 132 Effect of antisense oligonucleotide treatment on hemoglobin(g/dL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 13.2 12.9 12.4 13.2 12.713.0 12.8 day −5 13.1 13.1 12.7 13.2 13.0 13.2 12.8 day 8 13.1 12.9 12.412.8 12.7 12.8 12.5 day 15 12.9 12.9 12.1 12.6 12.8 12.3 12.2 day 2212.7 12.5 11.6 12.4 12.1 12.1 11.7 day 29 12.8 12.4 11.5 12.3 12.1 12.012.0 day 36 13.0 12.8 12.2 12.6 12.5 12.5 12.3 day 43 12.9 12.7 11.812.4 12.2 12.3 11.8 day 50 12.6 12.3 11.8 12.2 12.1 12.3 11.9 day 5713.1 12.6 12.1 12.7 12.3 11.3 n.d. day 64 13.1 12.6 12.3 12.8 12.1 12.2n.d. day 71 12.9 12.7 12.3 12.7 12.2 12.5 n.d. day 78 13.0 12.5 12.212.4 11.9 12.4 n.d. day 85 13.2 12.4 12.7 11.9 12.3 12.2 n.d. n.d. = nodata

TABLE 133 Effect of antisense oligonucleotide treatment on hematocrit(%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 46 42 41 43 43 44 44 day −544 42 43 42 44 45 43 day 8 44 43 43 43 44 44 43 day 15 44 42 40 40 42 4040 day 22 45 43 41 41 42 41 40 day 29 46 43 41 41 43 42 42 day 36 46 4342 40 42 42 41 day 43 46 43 40 40 42 41 40 day 50 48 44 42 41 44 43 42day 57 46 43 42 41 42 38 n.d. day 64 47 44 43 42 42 41 n.d. day 71 46 4443 42 44 43 n.d. day 78 43 41 41 39 39 40 n.d. day 85 43 42 42 39 40 41n.d. n.d. = no data

TABLE 134 Effect of antisense oligonucleotide treatment on MCV (fL) incynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850416858 416864 417002 416850* day −14 81 77 78 77 79 79 81 day −5 78 7677 75 79 80 78 day 8 77 77 80 77 78 79 79 day 15 78 75 76 74 74 76 75day 22 84 80 83 77 79 79 79 day 29 83 81 83 78 80 81 82 day 36 81 78 8075 76 78 76 day 43 80 78 79 74 77 77 77 day 50 84 80 83 76 79 80 80 day57 82 79 80 74 77 80 n.d. day 64 81 79 79 73 75 76 n.d. day 71 84 80 8075 79 78 n.d. day 78 78 76 79 72 74 75 n.d. day 85 77 77 77 72 74 76n.d. n.d. = no data

TABLE 135 Effect of antisense oligonucleotide treatment on MCH (pg) incynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850416858 416864 417002 416850* day −14 23 23 23 24 23 24 24 day −5 23 2323 23 23 24 23 day 8 23 23 23 23 23 23 23 day 15 23 23 23 23 23 23 23day 22 23 23 24 24 23 23 23 day 29 23 23 23 23 23 23 23 day 36 23 23 2323 23 23 23 day 43 23 23 23 23 22 23 23 day 50 22 23 23 23 22 23 23 day57 23 23 23 22 23 23 n.d. Day 64 23 23 22 22 23 22 n.d. Day 71 23 23 2322 23 23 n.d. Day 78 23 23 23 23 23 23 n.d. Day 85 23 23 22 22 23 23n.d. n.d. = no data

TABLE 136 Effect of antisense oligonucleotide treatment on MCHC (g/dL)in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850416858 416864 417002 416850* day −14 29 30 30 31 29 30 29 day −5 30 3130 31 29 30 30 day 8 30 30 29 30 29 29 29 day 15 30 31 30 31 30 31 30day 22 28 29 28 30 29 29 29 day 29 28 29 28 30 29 29 28 day 36 28 30 2931 30 30 30 day 43 28 30 29 31 29 30 30 day 50 26 28 28 30 28 29 29 day57 29 29 29 31 29 29 n.d. day 64 28 29 29 30 29 30 n.d. day 71 28 29 2830 28 29 n.d. day 78 30 30 29 32 30 31 n.d. day 85 31 30 30 31 30 30n.d. n.d. = no data

TABLE 137 Effect of antisense oligonucleotide treatment on plateletcount (× 10³/μL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS416838 416850 416858 416864 417002 416850* day −14 349 377 528 419 434442 387 day −5 405 425 573 463 456 466 434 day 8 365 387 548 391 438 435401 day 15 375 387 559 400 439 410 396 day 22 294 319 466 316 364 377347 day 29 311 337 475 336 397 410 370 day 36 326 370 505 371 428 415379 day 43 336 365 490 342 351 393 391 day 50 379 372 487 331 419 389351 day 57 345 371 528 333 409 403 n.d. day 64 329 358 496 295 383 436n.d. day 71 322 365 465 286 394 490 n.d. day 78 309 348 449 262 366 432n.d. day 85 356 344 458 267 387 418 n.d. n.d. = no data

TABLE 138 Effect of antisense oligonucleotide treatment on reticulocytes(%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 1.4 1.0 1.7 1.0 0.9 0.9 1.1day −5 1.0 0.9 1.2 0.9 0.9 0.8 0.8 day 8 1.0 1.2 1.2 1.2 0.8 1.1 1.1 day15 1.5 1.2 1.9 1.6 0.8 1.1 1.0 day 22 1.2 1.2 1.9 1.3 0.9 1.2 1.0 day 291.6 1.6 2.5 1.5 1.3 1.6 1.4 day 36 1.7 1.6 2.2 1.6 1.3 1.3 1.3 day 431.3 1.2 1.6 1.3 1.1 1.1 1.0 day 50 1.6 1.6 2.7 1.5 1.3 1.6 1.2 day 571.8 1.5 2.0 1.4 1.0 4.6 n.d. day 64 1.3 1.3 1.7 1.0 0.8 1.3 n.d. day 711.6 1.3 1.8 1.3 1.0 1.3 n.d. day 78 1.5 1.4 1.8 1.2 1.2 1.3 n.d. day 851.5 1.5 2.3 1.3 1.5 1.4 n.d. n.d. = no data

TABLE 139 Effect of antisense oligonucleotide treatment on neutrophils(%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 40 36 49 37 53 43 48 day −537 35 52 46 51 43 53 day 8 54 42 57 51 52 46 53 day 15 49 43 58 54 59 5773 day 22 41 37 57 47 59 55 64 day 29 44 36 53 43 44 45 42 day 36 37 3957 47 58 61 72 day 43 40 30 50 45 57 57 61 day 50 36 31 45 46 49 61 62day 57 41 32 49 44 57 54 n.d. day 64 40 30 41 37 49 55 n.d. day 71 38 2827 26 42 34 n.d. day 78 42 35 42 39 48 51 n.d. day 85 30 22 60 40 39 36n.d. n.d. = no data

TABLE 140 Effect of antisense oligonucleotide treatment on lymphocytes(%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 54 59 47 58 42 53 47 day −556 59 43 49 44 53 43 day 8 43 54 39 45 45 50 44 day 15 47 53 38 43 38 4024 day 22 54 59 39 49 37 41 33 day 29 51 59 43 51 51 50 53 day 36 58 5739 49 38 35 26 day 43 55 65 45 51 39 39 36 day 50 59 64 49 48 46 34 35day 57 55 63 45 51 39 40 n.d. day 64 56 64 53 56 46 39 n.d. day 71 56 6561 66 52 59 n.d. day 78 53 60 51 54 46 41 n.d. day 85 63 72 34 52 54 56n.d. n.d. = no data

TABLE 141 Effect of antisense oligonucleotide treatment on eosinophils(%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838416850 416858 416864 417002 416850* day −14 1.3 0.6 1.0 0.7 1.0 0.3 0.5day −5 1.5 0.6 1.6 1.3 0.9 0.3 0.7 day 8 0.9 0.4 1.1 0.3 0.7 0.2 0.5 day15 0.7 0.3 1.0 0.3 0.5 0.1 0.2 day 22 0.9 0.5 0.7 0.6 0.9 0.3 0.5 day 290.9 0.3 1.2 0.6 0.9 0.3 0.8 day 36 0.9 0.5 1.7 0.4 0.6 0.2 0.4 day 430.9 0.6 1.2 0.3 0.6 0.2 0.4 day 50 1.2 0.8 1.2 0.4 0.7 0.1 0.3 day 570.7 0.6 1.0 0.3 0.4 0.2 n.d. day 64 1.0 0.7 1.3 0.4 0.7 0.2 n.d. day 711.6 0.8 1.8 0.9 1.1 0.3 n.d. day 78 1.0 0.9 1.0 0.5 1.2 0.1 n.d. day 851.3 1.5 1.2 0.6 1.6 0.2 n.d. n.d. = no data

TABLE 142 Effect of antisense oligonucleotide treatment on monocytes (%)in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850416858 416864 417002 416850* day −14 3.3 3.1 2.3 2.8 2.8 3.0 2.9 day −53.8 3.6 2.8 2.8 3.3 3.2 2.4 day 8 2.3 2.5 1.8 2.7 2.1 3.3 1.8 day 15 2.72.4 2.0 2.2 2.4 2.3 1.5 day 22 3.4 2.9 2.4 2.8 2.8 3.1 1.9 day 29 3.33.2 2.7 3.8 3.4 3.5 2.7 day 36 3.1 2.5 2.1 2.9 2.3 2.6 1.5 day 43 3.53.3 2.6 3.1 2.1 2.8 1.8 day 50 2.6 3.2 3.7 4.6 2.9 3.1 1.8 day 57 2.63.2 n.d.3.2 3.8 2.4 3.6 n.d. day 64 2.6 3.5 n.d.3.5 4.4 2.8 4.0 n.d. day71 3.4 4.3 n.d.4.7 4.9 3.7 4.7 n.d. day 78 3.3 3.6 n.d.4.5 4.9 3.7 4.7n.d. day 85 4.4 3.7 n.d.3.5 6.1 3.7 5.3 n.d. n.d. = no data

TABLE 143 Effect of antisense oligonucleotide treatment on basophils (%)in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850416858 416864 417002 416850* day −14 0.3 0.2 0.2 0.3 0.2 0.3 0.2 day −50.3 0.3 0.2 0.3 0.2 0.3 0.3 day 8 0.2 0.2 0.2 0.3 0.2 0.3 0.3 day 15 0.30.3 0.2 0.2 0.2 0.2 0.2 day 22 0.2 0.2 0.2 0.2 0.2 0.2 0.1 day 29 0.30.2 0.2 0.2 0.3 0.2 0.3 day 36 0.3 0.4 0.3 0.3 0.3 0.2 0.1 day 43 0.30.4 0.3 0.3 0.4 0.3 0.2 day 50 0.4 0.3 0.3 0.4 0.4 0.3 0.2 day 57 0.20.3 0.4 0.2 0.3 0.3 n.d. day 64 0.3 0.4 0.4 0.4 0.4 0.2 n.d. day 71 0.20.5 0.3 0.4 0.4 0.3 n.d. day 78 0.2 0.4 0.3 0.4 0.3 0.3 n.d. day 85 0.30.3 0.3 0.3 0.4 0.3 n.d. n.d. = no data

Cytokine and Chemokine Assays

Blood samples obtained from the monkey groups treated with PBS, ISIS416850 and ISIS 416858 administered in the escalating dose regimen weresent to Pierce Biotechnology (Woburn, Mass.) for measurement ofchemokine and cytokine levels. Levels of IL-1β, IL-6, IFN-γ, and TNF-αwere measured using the respective primate antibodies and levels ofIL-8, MIP-1α, MCP-1, MIP-1β and RANTES were measured using therespective cross-reacting human antibodies. Measurements were taken 14days before the start of treatment and on day 85, when the monkeys wereeuthanized. The results are presented in Tables 144 and 145.

TABLE 144 Effect of antisense oligonucleotide treatment oncytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day −14 IL-1βIL-6 IFN-γ TNF-α IL-8 MIP-1α MCP-1 MIP-1β RANTES PBS 16 10 114 7 816 541015 118 72423 ISIS 416850 3 30 126 14 1659 28 1384 137 75335 ISIS416858 5 9 60 9 1552 36 1252 122 112253

TABLE 145 Effect of antisense oligonucleotide treatment oncytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day 85 IL-1βIL-6 IFN-γ TNF-α IL-8 MIP-1α MCP-1 MIP-1β RANTES PBS 7 4 102 34 87 23442 74 84430 ISIS 416850 13 17 18 27 172 41 2330 216 83981 ISIS 416858 525 18 45 303 41 1752 221 125511

Example 46: Pharmacologic Effect of Antisense Oligonucleotides TargetingHuman Factor 11 in Cynomolgus Monkeys

Several antisense oligonucleotides chosen from the rodent tolerabilitystudies (Examples 41-44) were tested in cynomolgus monkeys to determinetheir pharmacologic effects, relative efficacy on Factor 11 activity andtolerability in a cynomolgus monkey model. The antisenseoligonucleotides were also compared to ISIS 416850 and ISIS 416858selected from the monkey study described earlier (Example 45). All theISIS oligonucleotides used in this study target human Factor 11 mRNA andare also fully cross-reactive with the rhesus monkey gene sequence (seeTables 44 and 46). It is expected that the rhesus monkey ISISoligonucleotides are fully cross-reactive with the cynomolgus monkeygene sequence as well. At the time the study was undertaken, thecynomolgus monkey genomic sequence was not available in the NationalCenter for Biotechnology Information (NCBI) database; therefore,cross-reactivity with the cynomolgus monkey gene sequence could not beconfirmed.

Treatment

Groups, each consisting of two male and two female monkeys, wereinjected subcutaneously with 25 mg/kg of ISIS 416850, ISIS 449709, ISIS445522, ISIS 449710, ISIS 449707, ISIS 449711, ISIS 449708, 416858, andISIS 445531. Antisense oligonucleotide was administered to the monkeysat 25 mg/kg three times per week for week 1 and 25 mg/kg twice per weekfor weeks 2 to 8. A control group, consisting of two male and two femalemonkeys was injected subcutaneously with PBS according to the samedosing regimen. Body weights were taken 14 days and 7 days before thestart of treatment and were then measured weekly throughout thetreatment period. Blood samples were collected 14 days and 5 days beforethe start of treatment and subsequently several times during the dosingregimen for PT and aPTT measurements, and measurement of varioushematologic factors. On day 55, the monkeys were euthanized byexsanguination while under deep anesthesia, and organs harvested forfurther analysis.

RNA Analysis

On day 55, RNA was extracted from liver tissue for real-time PCRanalysis of Factor 11 using primer probe set LTS00301. Results arepresented as percent inhibition of Factor 11, relative to PBS control.As shown in Table 146, treatment with ISIS 416850, ISIS 449709, ISIS445522, ISIS 449710, ISIS 449707, ISIS 449708, ISIS 416858, and ISIS445531 resulted in significant reduction of Factor 11 mRNA in comparisonto the PBS control.

TABLE 146 Inhibition of Factor 11 mRNA in the cynomolgus monkey liverrelative to the PBS control % Oligo ID inhibition 416850 68 449709 69445522 89 449710 52 449707 47 449711 0 449708 46 416858 89 445531 66

Protein Analysis

Plasma samples from all monkey groups taken on different days wereanalyzed by a sandwich-style ELISA assay (Affinity Biologicals Inc.)using an affinity-purified polyclonal anti-Factor 11 antibody as thecapture antibody and a peroxidase-conjugated polyclonal anti-Factor 11antibody as the detecting antibody. Monkey plasma was diluted 1:50 forthe assay. Peroxidase activity was expressed by incubation with thesubstrate o-phenylenediamine. The color produced was quantified using amicroplate reader at 490 nm and was considered to be proportional to theconcentration of Factor 11 in the samples.

The results are presented in Table 147, expressed as percentagereduction relative to that of the PBS control. Treatment with ISIS416850, ISIS 449709, ISIS 445522, and ISIS 416858 resulted in atime-dependent decrease in protein levels.

TABLE 147 Inhibition of Factor 11 protein in the cynomolgus monkey liverrelative to the PBS control ISIS Day Day Day Day Day Day Day Day Day DayNo. −14 −5 10 17 24 31 38 45 52 55 416850 0 0 20 31 38 52 51 53 53 58449709 1 0 27 35 44 45 46 48 47 50 445522 2 0 36 50 61 70 73 77 80 82449710 1 0 10 14 17 25 20 23 4 24 449707 0 0 16 19 21 29 28 35 29 32449711 0 1 5 3 6 9 2 4 3 5 449708 1 0 7 15 3 14 9 2 6 6 416858 4 0 36 4962 68 74 79 81 81 445531 0 1 9 22 23 27 29 32 32 37PT and aPTT Assay

PT and aPTT were measured using platelet poor plasma (PPP) from micetreated with ISIS oligonucleotides. PT and aPTT values are provided inTables 148 and 149 and are reported as International Normalized Ratio(INR) values. INR values for PT and aPTT were determined by dividing thePT or aPTT value for each experimental group by the PT or aPTT for thePBS treated group. This ratio was then raised to the power of theInternational Sensitivity Index (ISI) of the tissue factor used. Asshown in Table 148, PT was not significantly prolonged in mice treatedwith ISIS oligonucleotides. However, aPTT was significantly prolonged ingroups treated with ISIS 416850, ISIS 445522, and ISIS 416858, aspresented in Table 149. These data suggest that antisense reduction ofFactor 11 affects the contact activation pathway, but not the extrinsicpathway of blood coagulation. Therefore, antisense reduction of Factor11 with these ISIS oligonucleotides is useful for inhibiting theformation of a thrombus or clot in response to an abnormal vessel wall,but not in response to tissue injury.

TABLE 148 Effect of antisense oligonucleotide treatment on PT ratio incynomolgus monkeys Day- Day- Day Day Day Day Day Day Day Day 14 5 10 1724 31 38 45 52 55 ISIS 416850 1.02 1.00 0.99 1.00 0.97 1.00 1.01 1.001.02 1.07 ISIS 449709 1.00 0.96 0.95 0.95 0.95 0.95 0.97 0.97 0.99 1.03ISIS 445522 1.00 0.94 0.95 0.96 0.94 0.96 0.97 0.96 0.98 1.01 ISIS449710 1.03 0.96 0.98 1.00 0.97 0.98 0.99 0.97 0.98 1.06 ISIS 4497071.01 0.94 0.95 0.97 0.95 0.96 1.00 0.96 0.96 1.00 ISIS 449711 1.00 0.950.94 0.95 0.94 0.98 1.02 1.01 1.00 1.07 ISIS 449708 1.03 0.95 0.98 1.000.95 1.06 0.99 0.99 0.99 1.04 ISIS 416858 1.01 0.96 0.96 0.98 0.95 1.000.97 1.00 0.99 1.01 ISIS 445531 1.06 1.00 1.00 1.06 1.02 1.04 1.03 1.011.04 1.06

TABLE 149 Effect of antisense oligonucleotide treatment on aPTT ratio incynomolgus monkeys Day- Day- Day Day Day Day Day Day Day Day 14 5 10 1724 31 38 45 52 55 ISIS 416850 0.99 0.90 0.98 1.01 1.05 1.22 1.25 1.341.32 1.45 ISIS 449709 0.99 0.91 0.99 1.03 1.05 1.08 1.08 1.15 1.09 1.17ISIS 445522 0.96 0.91 1.06 1.10 1.14 1.25 1.32 1.39 1.39 1.42 ISIS449710 1.07 0.98 1.00 0.97 1.00 1.04 1.02 1.06 1.03 1.07 ISIS 4497070.90 0.87 0.92 0.94 0.93 0.95 0.99 1.00 0.99 1.04 ISIS 449711 0.94 0.960.92 0.90 0.92 0.89 0.93 0.94 0.92 0.96 ISIS 449708 1.07 1.01 1.06 1.051.01 1.09 1.06 1.06 1.08 1.11 ISIS 416858 1.03 0.96 1.07 1.13 1.21 1.321.41 1.49 1.53 1.61 ISIS 445531 1.00 0.89 0.95 1.05 1.00 1.07 1.06 1.131.15 1.19

Body and Organ Weights

Body weights of each group are given in Table 150 expressed in grams.The results indicate that treatment with the antisense oligonucleotidesdid not cause any adverse changes in the health of the animals, whichmay have resulted in a significant alteration in weight compared to thePBS control. Organ weights were taken after the animals were euthanizedon day 55, and livers, kidneys and spleens were harvested. The resultsare presented in Table 150 expressed as a percentage of the body weightand also show no significant alteration in weights compared to the PBScontrol, with the exception of ISIS 449711, which caused increase inspleen weight.

TABLE 150 Weekly measurements of body weights (g) of cynomolgus monkeysISIS ISIS ISIS ISIS ISIS ISIS ISIS ISIS ISIS Days PBS 416850 449709445522 449710 449707 449711 449708 416858 445531 −14 2069 2061 2044 20502097 2072 2049 2096 2073 2079 −7 2107 2074 2093 2042 2114 2083 2105 21632092 2092 1 2131 2083 2112 2047 2131 2107 2123 2130 2115 2125 8 21862072 2075 2094 2120 2088 2123 2148 2149 2119 15 2201 2147 2085 2092 21452120 2103 2125 2162 2109 22 2206 2139 2117 2114 2177 2142 2171 2110 21882143 29 2204 2159 2068 2125 2149 2155 2203 2095 2196 2148 36 2246 21362064 2121 2180 2158 2227 2100 2210 2191 43 2304 2186 2106 2142 2227 21972251 2125 2238 2233 50 2274 2143 2147 2127 2201 2185 2227 2076 2225 2197

TABLE 151 Organ weights (g) of cynomolgus monkeys aftera ntisenseoligonucleotide treatment Liver Spleen Kidney PBS 2.3 0.16 0.48 ISIS416850 2.5 0.17 0.51 ISIS 449709 2.6 0.21 0.57 ISIS 445522 2.6 0.23 0.55ISIS 449710 2.6 0.24 0.58 ISIS 449707 2.5 0.24 0.53 ISIS 449711 2.6 0.320.54 ISIS 449708 2.6 0.19 0.60 ISIS 416858 2.6 0.24 0.47 ISIS 445531 2.80.24 0.49

Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function,plasma concentrations of ALT and AST were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of alanine transaminase (ALT) and aspartatetransaminase (AST) were measured and the results are presented in Tables152 and 153 expressed in IU/L. Plasma levels of bilirubin were alsomeasured and results are presented in Table 154 expressed in mg/dL. Asobserved in Tables 152-154, there were no significant increases in anyof the liver metabolic markers after antisense oligonucleotidetreatment.

TABLE 152 Effect of antisense oligonucleotide treatment on ALT (IU/L) inthe liver of cynomolgus monkeys Day −14 Day −5 Day 31 Day 55 PBS 57 5553 57 ISIS 416850 48 42 45 55 ISIS 449709 73 77 65 102 ISIS 445522 43 4540 60 ISIS 449710 37 42 37 45 ISIS 449707 54 56 52 63 ISIS 449711 49 13748 54 ISIS 449708 48 54 44 46 ISIS 416858 43 66 46 58 ISIS 445531 84 7357 73

TABLE 153 Effect of antisense oligonucleotide treatment on AST (IU/L) inthe liver of cynomolgus monkeys Day −14 Day −5 Day 31 Day 55 PBS 65 4544 47 ISIS 416850 62 45 46 57 ISIS 449709 62 51 45 71 ISIS 445522 62 4746 79 ISIS 449710 52 38 37 64 ISIS 449707 64 53 50 52 ISIS 449711 58 7847 47 ISIS 449708 74 53 56 50 ISIS 416858 64 100 60 69 ISIS 445531 78 4647 49

TABLE 154 Effect of antisense oligonucleotide treatment on bilirubin(mg/dL) in the liver of cynomolgus monkeys Day −14 Day −5 Day 31 Day 55PBS 0.25 0.20 0.20 0.17 ISIS 416850 0.26 0.22 0.26 0.17 ISIS 449709 0.240.19 0.15 0.18 ISIS 445522 0.24 0.20 0.14 0.18 ISIS 449710 0.24 0.190.15 0.22 ISIS 449707 0.27 0.19 0.13 0.16 ISIS 449711 0.23 0.16 0.130.13 ISIS 449708 0.27 0.21 0.14 0.14 ISIS 416858 0.25 0.23 0.16 0.16ISIS 445531 0.22 0.18 0.13 0.11

Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function,urine samples were collected on different days. BUN levels were measuredat various time points using an automated clinical chemistry analyzer(Hitachi Olympus AU400e, Melville, N.Y.) and the results are presentedin Table 155. The ratio of urine protein to creatinine in urine samplesafter antisense oligonucleotide treatment was also calculated for day 49and results are presented in Table 156. As observed in Tables 155 and156, there were no significant increases in any of the kidney metabolicmarkers after antisense oligonucleotide treatment.

TABLE 155 Effect of antisense oligonucleotide treatment on BUN levels(mg/dL) in cynomolgus monkeys Day −14 Day −5 Day 31 Day 55 PBS 22 21 2222 ISIS 416850 24 23 21 26 ISIS 449709 22 21 20 28 ISIS 445522 23 22 2222 ISIS 449710 19 19 19 23 ISIS 449707 25 21 21 20 ISIS 449711 26 22 2023 ISIS 449708 25 23 23 23 ISIS 416858 25 24 23 24 ISIS 445531 22 18 2022

TABLE 156 Effect of antisense oligonucleotide treatment on urine proteinto creatinine ratio in cynomolgus monkeys Urine protein/creatinine ratioPBS 0.02 ISIS 416850 0.08 ISIS 449709 0.05 ISIS 445522 0.01 ISIS 4497100.00 ISIS 449707 0.03 ISIS 449711 0.01 ISIS 449708 0.00 ISIS 416858 0.05ISIS 445531 0.08

Hematology Assays

Blood obtained from all the monkey groups on different days were sent toKorea Institute of Toxicology (KIT) for HCT, MCV, MCH, and MCHCmeasurements, as well as measurements of the various blood cells, suchas WBC (neutrophils and monocytes), RBC and platelets, as well as totalhemoglobin content. The results are presented in Tables 157-166.

TABLE 157 Effect of antisense oligonucleotide treatment on HCT (%) incynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55 PBS 40 42 4343 41 40 ISIS 416850 41 44 42 42 42 40 ISIS 449709 41 42 43 42 41 40ISIS 445522 42 42 41 43 41 39 ISIS 449710 41 44 43 44 43 41 ISIS 44970740 43 42 43 43 42 ISIS 449711 41 41 42 39 39 38 ISIS 449708 41 44 44 4344 42 ISIS 416858 41 44 43 43 41 39 ISIS 445531 41 42 43 41 41 41

TABLE 158 Effect of antisense oligonucleotide treatment on plateletcount (×100/μL) in cynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45Day 55 PBS 361 441 352 329 356 408 ISIS 416850 462 517 467 507 453 396ISIS 449709 456 481 449 471 418 441 ISIS 445522 433 512 521 425 403 333ISIS 449710 411 463 382 422 313 360 ISIS 449707 383 464 408 408 424 399ISIS 449711 410 431 325 309 257 259 ISIS 449708 387 517 444 378 381 348ISIS 416858 369 433 358 289 287 257 ISIS 445531 379 416 380 376 345 319

TABLE 159 Effect of antisense oligonucleotide treatment on neutrophils(%) in cynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55 PBS81 84 75 75 91 118 ISIS 416850 88 109 95 100 85 108 ISIS 449709 73 10189 81 77 115 ISIS 445522 61 84 81 66 69 125 ISIS 449710 93 86 80 94 97132 ISIS 449707 85 106 80 89 89 98 ISIS 449711 64 71 52 58 45 70 ISIS449708 73 84 61 57 61 75 ISIS 416858 65 84 54 54 61 73 ISIS 445531 60 8085 116 93 91

TABLE 160 Effect of antisense oligonucleotide treatment on monocytes (%)in cynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55 PBS 1.92.8 3.1 2.8 3.9 2.2 ISIS 416850 1.9 2.9 3.2 3.7 3.8 3.4 ISIS 449709 4.02.0 3.0 2.8 3.6 3.4 ISIS 445522 2.1 2.3 3.6 3.9 4.4 3.0 ISIS 449710 1.32.0 2.5 2.4 3.4 1.6 ISIS 449707 1.3 2.3 3.2 4.2 4.0 4.8 ISIS 449711 1.22.3 5.9 6.9 7.6 7.8 ISIS 449708 1.7 2.6 5.4 5.8 7.0 6.2 ISIS 416858 2.02.7 4.0 4.7 4.6 4.6 ISIS 445531 1.3 2.2 3.4 4.1 4.4 4.1

TABLE 161 Effect of antisense oligonucleotide treatment on hemoglobincontent (g/dL) in cynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45Day 55 PBS 12.3 12.5 12.9 12.7 12.4 12.1 ISIS 416850 13.0 13.5 13.3 13.113.1 12.7 ISIS 449709 12.8 12.8 13.2 13.1 12.6 12.5 ISIS 445522 13.312.7 12.7 12.9 12.6 12.0 ISIS 449710 13.0 13.2 13.4 13.1 13.0 12.7 ISIS449707 12.7 12.8 12.7 12.7 12.9 12.6 ISIS 449711 12.7 12.7 12.5 11.811.5 11.3 ISIS 449708 13.0 13.2 13.5 13.0 13.3 13.0 ISIS 416858 12.813.0 13.0 12.8 12.3 12.0 ISIS 445531 12.6 12.6 12.7 12.3 12.0 12.1

TABLE 162 Effect of antisense oligonucleotide treatment on WBC count(×10³/μL) in cynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55PBS 10 10 11 12 11 12 ISIS 416850 12 13 11 12 12 10 ISIS 449709 11 10 1111 11 10 ISIS 445522 10  9 11 13 10 11 ISIS 449710 11 11 12 12 11 15ISIS 449707 13 11 12 11 12  8 ISIS 449711 13 12 10  9  9  7 ISIS 44970814 10 11 11 10 10 ISIS 416858 10 11 10  9  8  9 ISIS 445531 20 15 17 1720 15

TABLE 163 Effect of antisense oligonucleotide treatment on RBC count(×10⁶/μL) in cynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55PBS 5.6 5.6 5.8 5.8 5.6 5.5 ISIS 416850 5.5 5.7 5.6 5.6 5.7 5.6 ISIS449709 5.8 5.8 5.9 5.9 5.7 5.7 ISIS 445522 5.9 5.6 5.6 5.8 5.7 5.4 ISIS449710 5.6 5.8 5.8 5.8 5.7 5.6 ISIS 449707 5.7 5.8 5.7 5.7 5.9 5.8 ISIS449711 5.6 5.7 5.6 5.4 5.4 5.3 ISIS 449708 5.7 5.9 5.9 5.8 6.0 5.8 ISIS416858 5.5 5.5 5.6 5.6 5.5 5.3 ISIS 445531 5.7 5.7 5.8 5.6 5.5 5.6

TABLE 164 Effect of antisense oligonucleotide treatment on MCV (fL) incynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55 PBS 72 74 7573 73 73 ISIS 416850 74 77 76 75 75 73 ISIS 449709 72 74 73 73 71 71ISIS 445522 72 74 74 75 73 72 ISIS 449710 75 77 75 75 75 73 ISIS 44970771 75 74 74 73 73 ISIS 449711 73 74 75 73 73 73 ISIS 449708 73 75 75 7574 74 ISIS 416858 75 79 78 76 75 75 ISIS 445531 72 74 75 75 75 74

TABLE 165 Effect of antisense oligonucleotide treatment on MCH (pg) incynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55 PBS 22.122.4 22.3 22.1 22.0 22.0 ISIS 416850 23.7 23.7 23.7 23.3 22.7 22.9 ISIS449709 22.4 22.3 22.5 22.2 21.0 22.0 ISIS 445522 22.6 22.5 22.8 22.422.4 22.2 ISIS 449710 23.0 22.8 23.1 22.6 21.8 22.7 ISIS 449707 22.222.2 22.1 22.1 22.6 21.9 ISIS 449711 22.6 22.7 22.2 22.1 21.7 21.3 ISIS449708 22.9 22.7 22.9 22.7 22.2 22.5 ISIS 416858 23.2 23.5 23.1 23.022.2 22.8 ISIS 445531 22.2 22.2 22.1 22.0 21.6 21.7

TABLE 166 Effect of antisense oligonucleotide treatment on MCHC (g/dL)in cynomolgus monkeys Day-14 Day-5 Day 17 Day 31 Day 45 Day 55 PBS 30.830.0 30.1 29.9 30.3 30.2 ISIS 416850 32.0 30.7 31.3 31.0 31.0 30.9 ISIS449709 31.4 30.3 30.7 30.7 31.1 31.2 ISIS 445522 31.4 30.4 30.9 30.030.7 31.0 ISIS 449710 31.2 29.7 30.7 30.1 30.4 31.1 ISIS 449707 31.429.8 30.0 29.8 29.8 30.0 ISIS 449711 31.0 30.7 29.9 29.8 29.6 29.5 ISIS449708 31.4 30.2 30.7 29.9 30.6 31.8 ISIS 416858 31.1 29.8 29.9 31.030.3 30.4 ISIS 445531 30.9 30.0 29.5 29.7 29.0 29.6

Cytokine and Chemokine Assays

Blood samples obtained from all monkey groups were sent to PierceBiotechnology (Woburn, Mass.) for measurements of chemokine and cytokinelevels. Levels of IL-1β, IL-6, IFN-γ, and TNF-α were measured using therespective primate antibodies and levels of IL-8, MIP-1α, MCP-1, MIP-1βand RANTES were measured using the respective cross-reacting humanantibodies. Measurements were taken 14 days before the start oftreatment and on day 55, when the monkeys were euthanized. The resultsare presented in Tables 167 and 168.

TABLE 167 Effect of antisense oligonucleotide treatment oncytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day-14 IL-MIP- MCP- IL-1ß 6 IFN-γ TNF-α IL-8 1α 1 MIP-1ß RANTES PBS 350 3 314 3282 27 277 8 297 ISIS 416850 215 1 115 4 45 14 434 31 4560 ISIS 449409137 1 37 9 34 13 290 14 2471 ISIS 445522 188 5 172 16 32 22 297 27 3477ISIS 449710 271 7 1115 72 29 20 409 18 1215 ISIS 449707 115 1 34 6 10616 294 13 3014 ISIS 449711 79 2 29 6 156 20 264 24 3687 ISIS 449708 35 127 12 184 11 361 19 11666 ISIS 416858 103 0 32 4 224 11 328 37 6521 ISIS445531 101 2 68 9 83 25 317 22 7825

TABLE 168 Effect of antisense oligonucleotide treatment oncytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day 55 MIP-MCP- MIP- IL-1ß IL-6 IFN-γ TNF-α IL-8 1α 1 1ß RANTES PBS 453 3 232 19168 21 237 34 775 ISIS 416850 106 1 19 16 620 17 887 50 27503 ISIS 449409181 0 25 8 254 17 507 47 8958 ISIS 445522 341 2 83 18 100 22 592 6316154 ISIS 449710 286 2 176 26 348 27 474 53 22656 ISIS 449707 97 1 2416 48 12 264 49 1193 ISIS 449711 146 7 22 31 110 17 469 91 3029 ISIS449708 131 0 18 17 85 23 409 128 4561 ISIS 416858 28 1 9 15 167 11 51247 5925 ISIS 445531 155 1 15 16 293 12 339 84 5935

Example 47: Measurement of Viscosity of ISIS Antisense OligonucleotidesTargeting Human Factor 11

The viscosity of antisense oligonucleotides targeting human Factor 11was measured with the aim of screening out antisense oligonucleotideswhich have a viscosity more than 40 cP at a concentration of 165-185mg/mL.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μLof water was added and the antisense oligonucleotide was dissolved intosolution by heating the vial at 50° C. Part of (75 μL) the pre-heatedsample was pipetted to a micro-viscometer (Cambridge). The temperatureof the micro-viscometter was set to 25° C. and the viscosity of thesample was measured. Another part (20 μL) of the pre-heated sample waspipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UVinstrument). The results are presented in Table 169.

TABLE 169 Viscosity and concentration of ISIS antisense oligonucleotidestargeting human Factor 11 Viscosity Concentration ISIS No. (cP) (mg/mL)412223 8 163 412224 98 186 412225 >100 162 413481 23 144 413482 16. 172416848 6 158 416850 67 152 416851 26 187 416852 29 169 416856 18 175416858 10 166 416859 10 161 416860 >100 154 416861 14 110 416863 9 165416866 >100 166 416867 8 168 445498 21 157 445504 20 139 445505 9 155445509 >100 167 445513 34 167 445522 63 173 445522 58 174 445530 25 177445531 15 155 445531 20 179 449707 7 166 449708 9 188 449709 65 171449710 7 186 449711 6 209 451541 10 168

1-54. (canceled)
 55. A method of treating a thromboembolic condition ina patient, by prolonging activated partial thromboplastin time (aPTT),comprising administering to the individual an effective amount of aFactor XI inhibitor compound comprising at least one heterocyclic basemoiety, wherein aPTT is prolonged by at least 20%, wherein saidadministering does not significantly prolong prothrombin time (PT) inthe patient.
 56. The method of claim 55, wherein aPTT is prolonged in adose-dependent manner.
 57. The method of claim 55, whereby bleedingrisk, as determined by a bleed volume, is not increased when compared tountreated individuals.
 58. The method of claim 57, whereby bleedingrisk, as determined by a bleed volume, is increased not more than 3-foldwhen compared to untreated individuals.
 59. The method of claim 55,wherein the Factor XI inhibitor compound is co-administered with acompound selected from the group consisting of aspirin, clopidogrel,dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban,rivaroxaban, and LOVENOX.
 60. The method of claim 55, whereby PlateletFactor 4 (PF-4) is decreased.
 61. The method of claim 55, wherein saidadministration does not affect a decrease in platelet count of more than50% in the patient.